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Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting.

Gal-Yam EN, Jeong S, Tanay A, Egger G, Lee AS, Jones PA - PLoS Genet. (2006)

Bottom Line: The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states.We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site.Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350-base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350-base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.

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A Model Proposed for the Activation Process at the GRP78 Promoter(A) The TATA box is occupied to a high extent even before induction, probably by TBP but also other factors.(B-D) After stress induction the ERSEs are sequentially loaded, from E1 to E3 while each ERSE is occupied at a different level (indicated by the size of the circles).(E, F) Recruitment of factors to the TIS is followed by release of the transcription factors from the ERSEs.
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pgen-0020160-g006: A Model Proposed for the Activation Process at the GRP78 Promoter(A) The TATA box is occupied to a high extent even before induction, probably by TBP but also other factors.(B-D) After stress induction the ERSEs are sequentially loaded, from E1 to E3 while each ERSE is occupied at a different level (indicated by the size of the circles).(E, F) Recruitment of factors to the TIS is followed by release of the transcription factors from the ERSEs.

Mentions: To test how these proposed states correlate with the time-course data, we analyzed the enrichment/deprivation of the various clusters in the preinduction (t = 0 h), early induction (t = 0.5 h, 1 h, 6 h), and late induction (t = 16 h) time points. This analysis revealed an overrepresentation of cluster 1 in the 0-h time point (p < 10−5, hypergeometric test), of clusters 3 to 5 in the pooled early induction time points (p < 10−6), and of cluster 6 in the 16-h time point (p<0.01). We also detected a matching underrepresentation of clusters 3 and 5 in the 0-h time point, of cluster 1 in the early induction time points, and of cluster 3 in the 16-h time point. The combination of the clustering data and the temporal enrichment analysis suggests that the GRP78 core promoter switches between a small repertoire of states and enables us to determine the chronological order of these states, proposing a model for its transcriptional activation process (Figure 6). The possible mechanisms governing the transition between such states are discussed below.


Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting.

Gal-Yam EN, Jeong S, Tanay A, Egger G, Lee AS, Jones PA - PLoS Genet. (2006)

A Model Proposed for the Activation Process at the GRP78 Promoter(A) The TATA box is occupied to a high extent even before induction, probably by TBP but also other factors.(B-D) After stress induction the ERSEs are sequentially loaded, from E1 to E3 while each ERSE is occupied at a different level (indicated by the size of the circles).(E, F) Recruitment of factors to the TIS is followed by release of the transcription factors from the ERSEs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1574359&req=5

pgen-0020160-g006: A Model Proposed for the Activation Process at the GRP78 Promoter(A) The TATA box is occupied to a high extent even before induction, probably by TBP but also other factors.(B-D) After stress induction the ERSEs are sequentially loaded, from E1 to E3 while each ERSE is occupied at a different level (indicated by the size of the circles).(E, F) Recruitment of factors to the TIS is followed by release of the transcription factors from the ERSEs.
Mentions: To test how these proposed states correlate with the time-course data, we analyzed the enrichment/deprivation of the various clusters in the preinduction (t = 0 h), early induction (t = 0.5 h, 1 h, 6 h), and late induction (t = 16 h) time points. This analysis revealed an overrepresentation of cluster 1 in the 0-h time point (p < 10−5, hypergeometric test), of clusters 3 to 5 in the pooled early induction time points (p < 10−6), and of cluster 6 in the 16-h time point (p<0.01). We also detected a matching underrepresentation of clusters 3 and 5 in the 0-h time point, of cluster 1 in the early induction time points, and of cluster 3 in the 16-h time point. The combination of the clustering data and the temporal enrichment analysis suggests that the GRP78 core promoter switches between a small repertoire of states and enables us to determine the chronological order of these states, proposing a model for its transcriptional activation process (Figure 6). The possible mechanisms governing the transition between such states are discussed below.

Bottom Line: The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states.We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site.Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350-base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350-base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.

Show MeSH