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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 Nfil3 activation shows rapid transcription but delayed mRNA maturation.(A) Schematic of Nfil3 gene showing primer locations relative to the annotated TSS. Putative TSS and PolyA sites also shown. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500–1000 genome copies. Profiles are representative (n = 3–5) except for primer at 319 (n = 1). (C) Qualitative PCR analysis of pre-mRNA expression using Nfil3 ChIP primers. The number of source cells used to produce cDNA for each profile is shown at the far left. 2,100 cells corresponds to input cDNA from 12.5 ng of total RNA. Negative images are presented to distinguish PCR analysis of pre-mRNAs from Pol II ChIP data. (D) Accumulated Pol II ChIP data from 2 to 4 independent measurements. Error bars indicate SEM, except for points with only two values (thicker error bars indicate the difference from average). (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (F) Analysis of mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) directed at terminal exon sequence. (G) Comparative TaqMan qPCR analysis of oligo dT and random hexamer primed cDNA. Profiles shows early pre-mRNA synthesis relative to delayed formation of the polyadenylated mRNA. Replicated data presented on a linear Y-axis to accent the temporal divergence.
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pone.0134442.g004: Analysis of Nfil3 activation shows rapid transcription but delayed mRNA maturation.(A) Schematic of Nfil3 gene showing primer locations relative to the annotated TSS. Putative TSS and PolyA sites also shown. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500–1000 genome copies. Profiles are representative (n = 3–5) except for primer at 319 (n = 1). (C) Qualitative PCR analysis of pre-mRNA expression using Nfil3 ChIP primers. The number of source cells used to produce cDNA for each profile is shown at the far left. 2,100 cells corresponds to input cDNA from 12.5 ng of total RNA. Negative images are presented to distinguish PCR analysis of pre-mRNAs from Pol II ChIP data. (D) Accumulated Pol II ChIP data from 2 to 4 independent measurements. Error bars indicate SEM, except for points with only two values (thicker error bars indicate the difference from average). (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (F) Analysis of mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) directed at terminal exon sequence. (G) Comparative TaqMan qPCR analysis of oligo dT and random hexamer primed cDNA. Profiles shows early pre-mRNA synthesis relative to delayed formation of the polyadenylated mRNA. Replicated data presented on a linear Y-axis to accent the temporal divergence.

Mentions: E4bp4 protein (Nfil3) is a transcription factor encoded by a gene annotated as 15 kbp in length (Fig 4A) and involved in stress and immune responses as well as circadian rhythms [64]; all processes that may be regulated by α1aARs. Despite complications, analysis of Nfil3 activation revealed high transcription velocity coupled with cotranscriptional regulation that delayed expression of mature message. Problematically, Pol II ChIP showed both basal and activated density shifted downstream relative to the expected TSS (Fig 4B), with the later shown by accumulated measurements for each primer set (Fig 4D). Subsequent analysis of pre-mRNA expression using ChIP primers demonstrated a nearly complete absence of pre-RNA synthesis immediately downstream of the expected start site (Fig 4C: 319, 344 bp). However, a recently predicted mRNA variant (X1) initiates downstream (2183 bp) and could explain both the low level of upstream Pol II (-614 bp) and the range of proximal density common to basal and activated Pol II (319–3481 bp). Both increased Pol II (Fig 4B) and pre-mRNA (Fig 4C) suggest promoter escape by 5 minutes and arrival at the 3’ terminus by 7 minutes, consistent with qPCR analysis directed at sequence within the final exon that also shows a small increase at 7 minutes (Fig 4F). Together, these results demonstrate the initial wave of Pol II displayed rapid transcriptional velocity near 4,000 bp/min. Further complicating analysis, none of the 3’ primer pairs (15089, 17410, 18054 bp) were part of the annotated Nfil3 mRNA, nevertheless, expression of these target sequences was robust (Fig 4C) and the profiles matched qPCR analysis directed at the terminal exon (Fig 4F), suggesting these sequences have been incorporated into mature messages. Of interest, pre-mRNA analysis (Fig 4C) showed a faint “pre” wave of transcription, which was proximal at 2 minutes (2426, 3481 bp) and more distal at 3 minutes (7233–17410 bp) followed in each case by a reduction in pre-mRNA levels.


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 Nfil3 activation shows rapid transcription but delayed mRNA maturation.(A) Schematic of Nfil3 gene showing primer locations relative to the annotated TSS. Putative TSS and PolyA sites also shown. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500–1000 genome copies. Profiles are representative (n = 3–5) except for primer at 319 (n = 1). (C) Qualitative PCR analysis of pre-mRNA expression using Nfil3 ChIP primers. The number of source cells used to produce cDNA for each profile is shown at the far left. 2,100 cells corresponds to input cDNA from 12.5 ng of total RNA. Negative images are presented to distinguish PCR analysis of pre-mRNAs from Pol II ChIP data. (D) Accumulated Pol II ChIP data from 2 to 4 independent measurements. Error bars indicate SEM, except for points with only two values (thicker error bars indicate the difference from average). (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (F) Analysis of mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) directed at terminal exon sequence. (G) Comparative TaqMan qPCR analysis of oligo dT and random hexamer primed cDNA. Profiles shows early pre-mRNA synthesis relative to delayed formation of the polyadenylated mRNA. Replicated data presented on a linear Y-axis to accent the temporal divergence.
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Related In: Results  -  Collection

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pone.0134442.g004: Analysis of Nfil3 activation shows rapid transcription but delayed mRNA maturation.(A) Schematic of Nfil3 gene showing primer locations relative to the annotated TSS. Putative TSS and PolyA sites also shown. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500–1000 genome copies. Profiles are representative (n = 3–5) except for primer at 319 (n = 1). (C) Qualitative PCR analysis of pre-mRNA expression using Nfil3 ChIP primers. The number of source cells used to produce cDNA for each profile is shown at the far left. 2,100 cells corresponds to input cDNA from 12.5 ng of total RNA. Negative images are presented to distinguish PCR analysis of pre-mRNAs from Pol II ChIP data. (D) Accumulated Pol II ChIP data from 2 to 4 independent measurements. Error bars indicate SEM, except for points with only two values (thicker error bars indicate the difference from average). (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (F) Analysis of mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) directed at terminal exon sequence. (G) Comparative TaqMan qPCR analysis of oligo dT and random hexamer primed cDNA. Profiles shows early pre-mRNA synthesis relative to delayed formation of the polyadenylated mRNA. Replicated data presented on a linear Y-axis to accent the temporal divergence.
Mentions: E4bp4 protein (Nfil3) is a transcription factor encoded by a gene annotated as 15 kbp in length (Fig 4A) and involved in stress and immune responses as well as circadian rhythms [64]; all processes that may be regulated by α1aARs. Despite complications, analysis of Nfil3 activation revealed high transcription velocity coupled with cotranscriptional regulation that delayed expression of mature message. Problematically, Pol II ChIP showed both basal and activated density shifted downstream relative to the expected TSS (Fig 4B), with the later shown by accumulated measurements for each primer set (Fig 4D). Subsequent analysis of pre-mRNA expression using ChIP primers demonstrated a nearly complete absence of pre-RNA synthesis immediately downstream of the expected start site (Fig 4C: 319, 344 bp). However, a recently predicted mRNA variant (X1) initiates downstream (2183 bp) and could explain both the low level of upstream Pol II (-614 bp) and the range of proximal density common to basal and activated Pol II (319–3481 bp). Both increased Pol II (Fig 4B) and pre-mRNA (Fig 4C) suggest promoter escape by 5 minutes and arrival at the 3’ terminus by 7 minutes, consistent with qPCR analysis directed at sequence within the final exon that also shows a small increase at 7 minutes (Fig 4F). Together, these results demonstrate the initial wave of Pol II displayed rapid transcriptional velocity near 4,000 bp/min. Further complicating analysis, none of the 3’ primer pairs (15089, 17410, 18054 bp) were part of the annotated Nfil3 mRNA, nevertheless, expression of these target sequences was robust (Fig 4C) and the profiles matched qPCR analysis directed at the terminal exon (Fig 4F), suggesting these sequences have been incorporated into mature messages. Of interest, pre-mRNA analysis (Fig 4C) showed a faint “pre” wave of transcription, which was proximal at 2 minutes (2426, 3481 bp) and more distal at 3 minutes (7233–17410 bp) followed in each case by a reduction in pre-mRNA levels.

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.