Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function

Abstract

Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor occupancy, but give no indication of kinetics and are poor predictors of transcription factor function at a given locus. To measure transcription-factor-binding dynamics across the genome, we performed competition ChIP (refs 6, 7) with a sequence-specific Saccharomyces cerevisiae transcription factor, Rap1 (ref. 8). Rap1-binding dynamics and Rap1 occupancy were only weakly correlated (R(2) = 0.14), but binding dynamics were more strongly linked to function than occupancy. Long Rap1 residence was coupled to transcriptional activation, whereas fast binding turnover, which we refer to as 'treadmilling', was linked to low transcriptional output. Thus, DNA-binding events that seem identical by conventional ChIP may have different underlying modes of interaction that lead to opposing functional outcomes. We propose that transcription factor binding turnover is a major point of regulation in determining the functional consequences of transcription factor binding, and is mediated mainly by control of competition between transcription factors and nucleosomes. Our model predicts a clutch-like mechanism that rapidly engages a treadmilling transcription factor into a stable binding state, or vice versa, to modulate transcription factor function.

Figure 1. Development of transcription factor competition-ChIP in yeast

(a) Schematic of Rap1 competition-ChIP yeast strain. (b) Growth comparison of competition yeast strain to wild-type in inducing (2% Galactose) and non-inducing (2% Dextrose) conditions. (c) Western blot using an antibody against Rap1 (y-300). Strains containing only a Rap1-Myc or only Rap1-Flag copy are shown to the right to indicate the size of isoform-specific bands. Actin loading control below. (d) To estimate the dynamics of induction, the ratio of induced Rap1-Myc and constitutive Rap1-Flag protein is plotted. Data is from two technical replicates of two independent time course replicates. Error bars represent standard error.

Figure 2. Rap1-bound sites exhibit distinct replacement dynamics

(a) A Rap1 turnover experiment over a 30-kb region of chromosome II. Rap1 motifs and peaks are indicated. (b) Average log₂ Myc/Flag values for all Rap1 targets (red) increase relative to non-Rap1 targets (blue). (c) Rap1-Myc competes with Rap1-Flag for binding. Average single channel intensity for Rap1-Myc and Rap1-Flag for a single probe (id:CHR15FS000978891) in the promoter of TYE7/YOR344C shows the increase in Rap1-Myc is coincident with the loss of Rap1-Flag. (d) Total Rap1 occupancy does not change during the time-course. Average total Rap1 occupancy (log₂ Rap1 IP (y-300)/input z-score) at Rap1 targets at time 0 versus 60 minutes is plotted. (e) Average log₂ Myc/Flag values for the promoter of ribosomal protein gene RPL29B (red points). The model fit for the residence time parameter that best fits this data is shown (black line). (f) Colorimetric representation of log₂ Myc/Flag values for all 465 Rap1 targets, sorted by the initial (normalized) log₂ Myc/Flag value. (g) For each site in (f), the log₂ Myc/Flag value predicted by our residence model based on the calculated residence time. (h) Rap1 occupancy (time 0 z-score) vs. Rap1 residence for 465 Rap1 targets (R²= 0.14, 0.37 spearman rank correlation).

Figure 3. RNA Pol II recruitment, mRNA production, and histone acetyltransferase recruitment is associated with long Rap1 residence

(a-d) In the left panel, Rap1 residence time is plotted on the x-axis. In the right panel, Rap1 occupancy is plotted on the x-axis. In both panels, the following is plotted on the y-axis: (a) RNA Pol II occupancy, (b) mRNA/hr, (c) mRNA levels at time 0 and (d) Histone acetyltransferase Esa1 occupancy z-scores. r_s is the Spearman correlation value. (e) Colorimetric representation of Spearman rank correlation between various genomic data sets and Rap1 occupancy (left) and Rap1 residence (right), ordered by the magnitude of the absolute difference between the oc cupancy and residence correlations for each comparison. WCE; Whole cell extract, PBM; Protein binding microarray. Telomeric targets excluded from analysis (Supplemental Text).

Figure 4. Evidence for competition between Rap1 and nucleosomes

(a) Colorimetric representation of in vivo nucleosome occupancy centered on Rap1 binding motifs. Loci ordered by Rap1 residence time (top) or Rap1 occupancy (bottom). The total number of Rap1 targets in each group is shown in parentheses. To the right are plots of the average nucleosome occupancy for each group centered on the Rap1 motif. Targets with multiple Rap1 motifs are represented by one randomly chosen motif. (b) Same as (a) for in vitro nucleosome occupancy. (c) Histone H3 turnover vs. Rap1 residence for ribosomal protein genes (red) and other targets (blue). (d) The number of Rap1-nucleosome interactions detected within each Rap1 target peak boundary on a log₁₀ scale. (e) Relative change in nucleosome occupancy following Rap1 depletion centered on Rap1 motifs grouped residence. A value of zero represents no relative change in nucleosome occupancy. (f) in vitro Rap1 affinity for its cognate target as measured by Protein Binding Microarray (PBM) compared to Rap1 residence. Colors represent histone H4 occupancy z-scores (> −1.5 purple (high),< −1.5 green (low). (g) Top position weight matrix motifs discovered for Rap1 targets grouped by residence. The number of targets for each group is in parentheses. (h) All motifs from the top position weight matrix for each residence group are colored by their A/T (purple) or G/C (green) content at each motif base position. (i) Percentage of A/T content for the entire motif (blue), AA/AT/TA/TT at the 12^th and 13^th motif position (green), TT at the 12^th and 13^th position (purple) and GG/GC/CG/CC at the 12^th and 13^th position (red) for Rap1 targets grouped by residence and telomeric regions.