Chromatin decouples promoter threshold from dynamic range - PubMed (original) (raw)
. 2008 May 8;453(7192):246-50.
doi: 10.1038/nature06867. Epub 2008 Apr 16.
Affiliations
- PMID: 18418379
- PMCID: PMC2435410
- DOI: 10.1038/nature06867
Chromatin decouples promoter threshold from dynamic range
Felix H Lam et al. Nature. 2008.
Abstract
Chromatin influences gene expression by restricting access of DNA binding proteins to their cognate sites in the genome. Large-scale characterization of nucleosome positioning in Saccharomyces cerevisiae has revealed a stereotyped promoter organization in which a nucleosome-free region (NFR) is present within several hundred base pairs upstream of the translation start site. Many transcription factors bind within NFRs and nucleate chromatin remodelling events which then expose other cis-regulatory elements. However, it is not clear how transcription-factor binding and chromatin influence quantitative attributes of gene expression. Here we show that nucleosomes function largely to decouple the threshold of induction from dynamic range. With a series of variants of one promoter, we establish that the affinity of exposed binding sites is a primary determinant of the level of physiological stimulus necessary for substantial gene activation, and sites located within nucleosomal regions serve to scale expression once chromatin is remodelled. Furthermore, we find that the S. cerevisiae phosphate response (PHO) pathway exploits these promoter designs to tailor gene expression to different environmental phosphate levels. Our results suggest that the interplay of chromatin and binding-site affinity provides a mechanism for fine-tuning responses to the same cellular state. Moreover, these findings may be a starting point for more detailed models of eukaryotic transcriptional control.
Figures
Figure 1. PHO5 promoter variants and quantitative expression behaviour
a, Schematic of all PHO5 promoter variants controlling expression of yeGFP1. Large grey ovals represent nucleosomes, red triangles the high-affinity Pho4 motif (CACGTGg), blue ovals the low-affinity Pho4 motif (CACGTTt), and X the motif ablations. b, Physiological transcriptional response to extracellular inorganic phosphate (Pi) measured by flow cytometry. Data points represent median steady-state expression levels normalized to the median observed in Pi starvation. Error bars represent interquartile ranges, which were observed to encompass the medians of at least three independent measurements. c, Induction kinetics in Pi starvation. Data points represent median fluorescence levels scaled between the promoter-specific expression minimum at 0 h and maximum at 7 h. For b and c, red traces designate variants with an exposed high-affinity site, and blue traces variants with an exposed low-affinity site.
Figure 2. Promoter architecture and quantitative expression behaviour of representative PHO genes
a, b, Promoter architecture schematized by superimposing nucleosome positions measured in repressing (10 mM Pi) conditions onto Pho4-binding sites identified through bioinformatic analysis (Supplementary Fig. 4). Red triangles represent evolutionarily conserved high-affinity motifs (CACGTG consensus), dark-blue ovals represent evolutionarily conserved low-affinity motifs (deviations from the high-affinity motif), light-blue ovals represent low-affinity motifs that are not evolutionarily conserved (Supplementary Fig. 5), and the x axis units reference promoter coordinates with respect to translation start (ATG = 1). In a are PHO promoters with an accessible low-affinity Pho4 site; in b are promoters with at least one accessible high-affinity Pho4 site. c, Steady-state transcriptional response of PHO target genes to Pi. Error bars are interquartile ranges (see Fig. 1b). d, Induction kinetics in Pi starvation.
Figure 2. Promoter architecture and quantitative expression behaviour of representative PHO genes
a, b, Promoter architecture schematized by superimposing nucleosome positions measured in repressing (10 mM Pi) conditions onto Pho4-binding sites identified through bioinformatic analysis (Supplementary Fig. 4). Red triangles represent evolutionarily conserved high-affinity motifs (CACGTG consensus), dark-blue ovals represent evolutionarily conserved low-affinity motifs (deviations from the high-affinity motif), light-blue ovals represent low-affinity motifs that are not evolutionarily conserved (Supplementary Fig. 5), and the x axis units reference promoter coordinates with respect to translation start (ATG = 1). In a are PHO promoters with an accessible low-affinity Pho4 site; in b are promoters with at least one accessible high-affinity Pho4 site. c, Steady-state transcriptional response of PHO target genes to Pi. Error bars are interquartile ranges (see Fig. 1b). d, Induction kinetics in Pi starvation.
Figure 3. Maximum expression of PHO5 promoter variants
Maximal induction levels of PHO5 promoter variants measured from strains containing a deletion of the PHO80 gene. The dotted vertical line references the expression output of the wild-type promoter. Data points represent mean ± s.d. from triplicate measurements. a.u., arbitrary units.
Figure 4. Pho4 binding in vivo to PHO promoters, and model of threshold-dynamic range decoupling
a, Chromatin immunoprecipitation of Pho4 to PHO target genes and the H4 PHO5 promoter variant (see Fig. 1a) shows differential occupancy in intermediate Pi conditions at promoters with an exposed high- (H) versus low (L)-affinity site. Data points represent mean ± s.d. from at least three independent experiments. b, Schematic depicting a possible mechanism that decouples promoter induction threshold from dynamic range. In high Pi (top row), nucleosomes are fully assembled; in intermediate Pi (middle row), substantial Pho4 occupancy occurs only at promoters with exposed high-affinity sites, resulting in chromatin remodelling and transcription commensurate to the total Pho4 recruited; and in Pi starvation (bottom row), saturating Pho4 activity results in remodelling and maximum expression at all promoters. Nucleosome occupancy is indicated by the opacity of grey ovals, Pho4 by yellow ovals, and the amount of transcription by the thickness of the green arrows. See text for description.
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