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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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Few Combinatorial Modes of GRP78 Promoter OrganizationShown are clustered protection patterns for the 294 sampled promoters (rows, see Materials and Methods). Only few modes of promoter organization are observed, including clusters representing high levels of TATA binding (cluster 1), cassette like loading of the ESREs (clusters 2–4), recruitment of factors to the TIS (cluster 5), and release of the ESRE modules (cluster 6). Statistical enrichment analysis (Materials and Methods) confirms that specific modes of activity (clusters) are overrepresented in specific phases of the ER-stress activation process, enabling us to arrange the clusters in a chronological order. The designation of each row (= protection pattern of one promoter molecule) to the time point from which it originated is marked by the blue boxes on the right. The early induction time points (1, 0.5, and 6 h) are pooled.
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pgen-0020160-g005: Few Combinatorial Modes of GRP78 Promoter OrganizationShown are clustered protection patterns for the 294 sampled promoters (rows, see Materials and Methods). Only few modes of promoter organization are observed, including clusters representing high levels of TATA binding (cluster 1), cassette like loading of the ESREs (clusters 2–4), recruitment of factors to the TIS (cluster 5), and release of the ESRE modules (cluster 6). Statistical enrichment analysis (Materials and Methods) confirms that specific modes of activity (clusters) are overrepresented in specific phases of the ER-stress activation process, enabling us to arrange the clusters in a chronological order. The designation of each row (= protection pattern of one promoter molecule) to the time point from which it originated is marked by the blue boxes on the right. The early induction time points (1, 0.5, and 6 h) are pooled.

Mentions: The single molecule resolution afforded by M-SPA allowed us to examine possible linkage between the various footprint modules on the promoter. Looking at the correlation between nucleosomal region protection and transcription factors binding revealed that while at the population level stress induction resulted in an overall decrease of nucleosomal patches downstream of the TIS, at the single molecule level, no specific correlation existed between nucleosome positions and transcription factor binding (Figure 4B). In order to study the nucleosome-free region itself, we pooled data from all time points and performed an unsupervised clustering analysis of the protection patterns at CpGs 26 to 49, covering the TIS and the region upstream of it (Figure 5). Interestingly, all sequences were grouped into six defined clusters, suggesting that the GRP78 core promoter exists in six major modes and that its organization is controlled by strong structural or mechanistic constraints. Cluster 1 represents promoters of which most, but not all, are TATA box protected, probably by TBP and other factors. Clusters 2, 3, and 4 represent cassette-like loading of transcription factors on the ESREs. As there is almost complete lack of protection of ERSE2 without 1 or protection of ERSE3 without 1 and 2, this may reflect sequential loading of these sites (from ERSE1 to ERSE3) during induction. Cluster 5 represents recruitment of the transcriptional machinery to the TIS, and in cluster 6, the TIS is protected while ERSEs are not.


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)

Few Combinatorial Modes of GRP78 Promoter OrganizationShown are clustered protection patterns for the 294 sampled promoters (rows, see Materials and Methods). Only few modes of promoter organization are observed, including clusters representing high levels of TATA binding (cluster 1), cassette like loading of the ESREs (clusters 2–4), recruitment of factors to the TIS (cluster 5), and release of the ESRE modules (cluster 6). Statistical enrichment analysis (Materials and Methods) confirms that specific modes of activity (clusters) are overrepresented in specific phases of the ER-stress activation process, enabling us to arrange the clusters in a chronological order. The designation of each row (= protection pattern of one promoter molecule) to the time point from which it originated is marked by the blue boxes on the right. The early induction time points (1, 0.5, and 6 h) are pooled.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0020160-g005: Few Combinatorial Modes of GRP78 Promoter OrganizationShown are clustered protection patterns for the 294 sampled promoters (rows, see Materials and Methods). Only few modes of promoter organization are observed, including clusters representing high levels of TATA binding (cluster 1), cassette like loading of the ESREs (clusters 2–4), recruitment of factors to the TIS (cluster 5), and release of the ESRE modules (cluster 6). Statistical enrichment analysis (Materials and Methods) confirms that specific modes of activity (clusters) are overrepresented in specific phases of the ER-stress activation process, enabling us to arrange the clusters in a chronological order. The designation of each row (= protection pattern of one promoter molecule) to the time point from which it originated is marked by the blue boxes on the right. The early induction time points (1, 0.5, and 6 h) are pooled.
Mentions: The single molecule resolution afforded by M-SPA allowed us to examine possible linkage between the various footprint modules on the promoter. Looking at the correlation between nucleosomal region protection and transcription factors binding revealed that while at the population level stress induction resulted in an overall decrease of nucleosomal patches downstream of the TIS, at the single molecule level, no specific correlation existed between nucleosome positions and transcription factor binding (Figure 4B). In order to study the nucleosome-free region itself, we pooled data from all time points and performed an unsupervised clustering analysis of the protection patterns at CpGs 26 to 49, covering the TIS and the region upstream of it (Figure 5). Interestingly, all sequences were grouped into six defined clusters, suggesting that the GRP78 core promoter exists in six major modes and that its organization is controlled by strong structural or mechanistic constraints. Cluster 1 represents promoters of which most, but not all, are TATA box protected, probably by TBP and other factors. Clusters 2, 3, and 4 represent cassette-like loading of transcription factors on the ESREs. As there is almost complete lack of protection of ERSE2 without 1 or protection of ERSE3 without 1 and 2, this may reflect sequential loading of these sites (from ERSE1 to ERSE3) during induction. Cluster 5 represents recruitment of the transcriptional machinery to the TIS, and in cluster 6, the TIS is protected while ERSEs are not.

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