Limits...
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 Nr4a1 shows a distinct basal and two activated mRNAs.(A) Schematic of Nr4a1 gene including upstream TSSs. Primer locations are indicated relative to the dominant TSS. (B) Non-quantitative Pol II ChIP with high input DNA (2500 genome copies) suggests most basal transcriptional activity originates from the upstream TTS(b) location. Activated Pol II density shows signal saturation downstream of both the dominant TSS and second activated TSS(c). (C) Quantitative Pol II ChIP using input DNA from 500 genome copies. Pol II density from activated TSS(c) is within dynamic range. (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 (shown in next panel). (E) Analysis of relative Nr4a1 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) following excision of the terminal intron.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526373&req=5

pone.0134442.g003: Temporal Pol II ChIP analysis of Nr4a1 shows a distinct basal and two activated mRNAs.(A) Schematic of Nr4a1 gene including upstream TSSs. Primer locations are indicated relative to the dominant TSS. (B) Non-quantitative Pol II ChIP with high input DNA (2500 genome copies) suggests most basal transcriptional activity originates from the upstream TTS(b) location. Activated Pol II density shows signal saturation downstream of both the dominant TSS and second activated TSS(c). (C) Quantitative Pol II ChIP using input DNA from 500 genome copies. Pol II density from activated TSS(c) is within dynamic range. (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 (shown in next panel). (E) Analysis of relative Nr4a1 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) following excision of the terminal intron.

Mentions: A second member of the Nr4a family of stress response genes, Nr4a1, is also strongly upregulated by cardiac ischemia and α1aAR stimulation (S2 Table). Following agonist addition, Pol II ChIP revealed complex transcriptional behavior requiring many primer pairs at the positions indicated in the Nr4a1 gene schematic (Fig 3A). Non-quantitative analysis using 1/200 of standard ChIP (Fig 3B), shows no basal concentration of Pol II near any transcription start site, but does show modest levels of basal transcription, most of which appears to originate from a putative TSS(b) upstream of the dominant TSS (Fig 3A). Consistent with the position of TSS(b), this region is near the start site for a recently referenced rat gene (XM_006242358.2) and orthologous to conserved sequence containing a putative human TSS (i.e. NM_173158.1). At this ChIP input, signal saturation was apparent within 7 minutes of receptor stimulation on the dominant transcriptional unit and 11 kbp upstream (Fig 3B) near the location where the recent genome build [Rnor_6.0] annotates a TSS (-11644 bp). However, ChIP analysis using 5-fold less input DNA (Fig 3C) showed upstream signal to be less than that associated with the dominant TSS, which continues to display saturating signal (-893 to 9176 bp).


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 Nr4a1 shows a distinct basal and two activated mRNAs.(A) Schematic of Nr4a1 gene including upstream TSSs. Primer locations are indicated relative to the dominant TSS. (B) Non-quantitative Pol II ChIP with high input DNA (2500 genome copies) suggests most basal transcriptional activity originates from the upstream TTS(b) location. Activated Pol II density shows signal saturation downstream of both the dominant TSS and second activated TSS(c). (C) Quantitative Pol II ChIP using input DNA from 500 genome copies. Pol II density from activated TSS(c) is within dynamic range. (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 (shown in next panel). (E) Analysis of relative Nr4a1 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) following excision of the terminal intron.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134442.g003: Temporal Pol II ChIP analysis of Nr4a1 shows a distinct basal and two activated mRNAs.(A) Schematic of Nr4a1 gene including upstream TSSs. Primer locations are indicated relative to the dominant TSS. (B) Non-quantitative Pol II ChIP with high input DNA (2500 genome copies) suggests most basal transcriptional activity originates from the upstream TTS(b) location. Activated Pol II density shows signal saturation downstream of both the dominant TSS and second activated TSS(c). (C) Quantitative Pol II ChIP using input DNA from 500 genome copies. Pol II density from activated TSS(c) is within dynamic range. (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 (shown in next panel). (E) Analysis of relative Nr4a1 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) following excision of the terminal intron.
Mentions: A second member of the Nr4a family of stress response genes, Nr4a1, is also strongly upregulated by cardiac ischemia and α1aAR stimulation (S2 Table). Following agonist addition, Pol II ChIP revealed complex transcriptional behavior requiring many primer pairs at the positions indicated in the Nr4a1 gene schematic (Fig 3A). Non-quantitative analysis using 1/200 of standard ChIP (Fig 3B), shows no basal concentration of Pol II near any transcription start site, but does show modest levels of basal transcription, most of which appears to originate from a putative TSS(b) upstream of the dominant TSS (Fig 3A). Consistent with the position of TSS(b), this region is near the start site for a recently referenced rat gene (XM_006242358.2) and orthologous to conserved sequence containing a putative human TSS (i.e. NM_173158.1). At this ChIP input, signal saturation was apparent within 7 minutes of receptor stimulation on the dominant transcriptional unit and 11 kbp upstream (Fig 3B) near the location where the recent genome build [Rnor_6.0] annotates a TSS (-11644 bp). However, ChIP analysis using 5-fold less input DNA (Fig 3C) showed upstream signal to be less than that associated with the dominant TSS, which continues to display saturating signal (-893 to 9176 bp).

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.