Occlusion of Regulatory Sequences by Promoter Nucleosomes In Vivo

Nucleosomes are believed to inhibit DNA binding by transcription factors. Theoretical attempts to understand the significance of nucleosomes in gene expression and regulation are based upon this assumption. However, nucleosomal inhibition of transcription factor binding to DNA is not complete. Rather, access to nucleosomal DNA depends on a number of factors, including the stereochemistry of transcription factor-DNA interaction, the in vivo kinetics of thermal fluctuations in nucleosome structure, and the intracellular concentration of the transcription factor. In vitro binding studies must therefore be complemented with in vivo measurements. The inducible PHO5 promoter of yeast has played a prominent role in this discussion. It bears two binding sites for the transcriptional activator Pho4, which at the repressed promoter are positioned within a nucleosome and in the linker region between two nucleosomes, respectively. Earlier studies suggested that the nucleosomal binding site is inaccessible to Pho4 binding in the absence of chromatin remodeling. However, this notion has been challenged by several recent reports. We therefore have reanalyzed transcription factor binding to the PHO5 promoter in vivo, using ‘chromatin endogenous cleavage’ (ChEC). Our results unambiguously demonstrate that nucleosomes effectively interfere with the binding of Pho4 and other critical transcription factors to regulatory sequences of the PHO5 promoter. Our data furthermore suggest that Pho4 recruits the TATA box binding protein to the PHO5 promoter

Nucleosomal proofreading of activator–promoter interactions

Specificity in transcriptional regulation is imparted by transcriptional activators that bind to specific DNA sequences from which they stimulate transcription. Specificity may be increased by slowing down the kinetics of regulation: by increasing the energy for dissociation of the activator-DNA complex or decreasing activator concentration. In general, higher dissociation energies imply longer DNA dwell times of the activator; the activator-bound gene may not readily turn off again. Lower activator concentrations entail longer pauses between binding events; the activator-unbound gene is not easily turned on again and activated transcription occurs in stochastic bursts. We show that kinetic proofreading of activator-DNA recognition-insertion of an energy-dissipating delay step into the activation pathway for transcription-reconciles high specificity of transcriptional regulation with fast regulatory kinetics. We show that kinetic proofreading results from the stochastic removal and reformation of promoter nucleosomes, at a distance from equilibrium

Nucleosomal promoter variation generates gene expression noise

Gene product molecule numbers fluctuate over time and between cells, confounding deterministic expectations. The molecular origins of this noise of gene expression remain unknown. Recent EM analysis of single PHO5 gene molecules of yeast indicated that promoter molecules stochastically assume alternative nucleosome configurations at steady state, including the fully nucleosomal and nucleosome-free configuration. Given that distinct configurations are unequally conducive to transcription, the nucleosomal variation of promoter molecules may constitute a source of gene expression noise. This notion, however, implies an untested conjecture, namely that the nucleosomal variation arises de novo or intrinsically (i.e., that it cannot be explained as the result of the promoter's deterministic response to variation in its molecular surroundings). Here, we show--by microscopically analyzing the nucleosome configurations of two juxtaposed physically linked PHO5 promoter copies--that the configurational variation, indeed, is intrinsically stochastic and thus, a cause of gene expression noise rather than its effect

Nucleosomal promoter variation generates gene expression noise

Gene product molecule numbers fluctuate over time and between cells, confounding deterministic expectations. The molecular origins of this noise of gene expression remain unknown. Recent EM analysis of single PHO5 gene molecules of yeast indicated that promoter molecules stochastically assume alternative nucleosome configurations at steady state, including the fully nucleosomal and nucleosome-free configuration. Given that distinct configurations are unequally conducive to transcription, the nucleosomal variation of promoter molecules may constitute a source of gene expression noise. This notion, however, implies an untested conjecture, namely that the nucleosomal variation arises de novo or intrinsically (i.e., that it cannot be explained as the result of the promoter’s deterministic response to variation in its molecular surroundings). Here, we show—by microscopically analyzing the nucleosome configurations of two juxtaposed physically linked PHO5 promoter copies—that the configurational variation, indeed, is intrinsically stochastic and thus, a cause of gene expression noise rather than its effect

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms

Affinity Purification of Specific Chromatin Segments from Chromosomal Loci in Yeast

Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states