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

PLoS Genet. 2006 Sep 22;2(9):e160. doi: 10.1371/journal.pgen.0020160.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Pairing
  • Base Sequence
  • DNA Footprinting / methods*
  • DNA Modification Methylases / metabolism
  • Endoplasmic Reticulum / pathology
  • Gene Expression Regulation
  • Heat-Shock Proteins / genetics*
  • Humans
  • Kinetics
  • Models, Genetic
  • Molecular Chaperones / genetics*
  • Molecular Sequence Data
  • Nucleosomes / metabolism*
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transcription Factors / metabolism

Substances

  • Heat-Shock Proteins
  • Molecular Chaperones
  • Nucleosomes
  • RNA, Messenger
  • Transcription Factors
  • DNA Modification Methylases
  • molecular chaperone GRP78