Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting

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. Control of gene expression and transcription are complex and well-coordinated processes. Most current experimental approaches to understanding the underlying mechanisms, which include binding of transcription factors to regulatory regions of genes, and changes in the structure and composition of chromatin, rely on studies of populations of cells and cannot capture the transcription activation process on single promoters. The authors describe the use of a footprinting method which enables analysis of chromatin structure and binding of factors on single DNA molecules. This is applied to study the activation process of GRP78, a protein which is important for the induction of a response to endoplasmic reticulum stress. By combining the footprinting method and computational analyses, the authors define functional modules on the GRP78 promoter and show that it exists in few major combinatorial states, reflecting its high level of organization. These results provide novel insights into the activation of GRP78 which could not be gleaned using conventional methods. They also demonstrate the use of the method as a unique and powerful tool to study the transcriptional process in eukaryotes, which remains a major source of interest and challenge for the scientific community.