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.


Related in: MedlinePlus

Schematic illustrating the dominant regulatory events that control IEG expression.Together, Pol II ChIP and mRNA expression analysis can determine both the temporal effect and relative contribution of major transcriptional events to gene activation, including Pol II recruitment, promoter proximal pausing, internal and polyadenylation associated transcriptional blocks and control of mRNA stability. Gene names indicate events regulated during activation of the indicated gene.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134442.g007: Schematic illustrating the dominant regulatory events that control IEG expression.Together, Pol II ChIP and mRNA expression analysis can determine both the temporal effect and relative contribution of major transcriptional events to gene activation, including Pol II recruitment, promoter proximal pausing, internal and polyadenylation associated transcriptional blocks and control of mRNA stability. Gene names indicate events regulated during activation of the indicated gene.

Mentions: Based on a limited set of genes, analysis of IEG expression using Pol II ChIP and mRNA analysis suggests gene regulation will often involve both recruitment and post-recruitment mechanisms. This analysis was able to distinguish increased recruitment of Pol II and regulation of mRNA stability from co-transcriptionally regulated mechanisms including: abrogation of promoter proximal pausing, intragenic transcriptional blocks, and delayed mRNA maturation due to polyadenylation associated mRNA degradation (Fig 7). Even though co-transcriptional regulatory mechanisms were present for 5/6 of the genes analyzed, increased recruitment was a substantial factor contributing to increased mRNA levels for all six genes. Consequently, simple release of lower levels of basal Pol II from promoter proximal regions could not have induced full gene activation. Techniques for measuring genetic mechanisms (Fig 7) are generally performed under different assay conditions and are not quantitatively comparable, thus it is difficult to use these methods to infer the relative importance of the individual mechanisms. As this information is essential to delineating the role of specific cell signaling pathways in activating particular genetic mechanisms, current understanding of the process employed by these signaling pathways to integrate genetic mechanisms is necessarily incomplete. For IEG activation during severe stress, the time required to complete the transcriptional process is also important, as cells must race to produce mature mRNAs in the face of deteriorating energetic situations [69] and translational shutdown [69, 70]. Integrated temporal analysis simultaneously addresses the relative importance of major mechanisms of gene regulation (Fig 7) both temporally and quantitatively. Although long IEG genes could possess unusual characteristics, temporal analysis of these six genes displayed a broad range of regulatory mechanisms that may be of general relevance.


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)

Schematic illustrating the dominant regulatory events that control IEG expression.Together, Pol II ChIP and mRNA expression analysis can determine both the temporal effect and relative contribution of major transcriptional events to gene activation, including Pol II recruitment, promoter proximal pausing, internal and polyadenylation associated transcriptional blocks and control of mRNA stability. Gene names indicate events regulated during activation of the indicated gene.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134442.g007: Schematic illustrating the dominant regulatory events that control IEG expression.Together, Pol II ChIP and mRNA expression analysis can determine both the temporal effect and relative contribution of major transcriptional events to gene activation, including Pol II recruitment, promoter proximal pausing, internal and polyadenylation associated transcriptional blocks and control of mRNA stability. Gene names indicate events regulated during activation of the indicated gene.
Mentions: Based on a limited set of genes, analysis of IEG expression using Pol II ChIP and mRNA analysis suggests gene regulation will often involve both recruitment and post-recruitment mechanisms. This analysis was able to distinguish increased recruitment of Pol II and regulation of mRNA stability from co-transcriptionally regulated mechanisms including: abrogation of promoter proximal pausing, intragenic transcriptional blocks, and delayed mRNA maturation due to polyadenylation associated mRNA degradation (Fig 7). Even though co-transcriptional regulatory mechanisms were present for 5/6 of the genes analyzed, increased recruitment was a substantial factor contributing to increased mRNA levels for all six genes. Consequently, simple release of lower levels of basal Pol II from promoter proximal regions could not have induced full gene activation. Techniques for measuring genetic mechanisms (Fig 7) are generally performed under different assay conditions and are not quantitatively comparable, thus it is difficult to use these methods to infer the relative importance of the individual mechanisms. As this information is essential to delineating the role of specific cell signaling pathways in activating particular genetic mechanisms, current understanding of the process employed by these signaling pathways to integrate genetic mechanisms is necessarily incomplete. For IEG activation during severe stress, the time required to complete the transcriptional process is also important, as cells must race to produce mature mRNAs in the face of deteriorating energetic situations [69] and translational shutdown [69, 70]. Integrated temporal analysis simultaneously addresses the relative importance of major mechanisms of gene regulation (Fig 7) both temporally and quantitatively. Although long IEG genes could possess unusual characteristics, temporal analysis of these six genes displayed a broad range of regulatory mechanisms that may be of general relevance.

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.


Related in: MedlinePlus