Single-Molecule Analysis Reveals Linked Cycles of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast

Abstract

It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.

Keywords: ACE1; CUP1; RSC complex; Saccharomyces cerevisiae; chromatin remodeling; single molecule tracking; smFISH; transcription bursts; transcription factors; transient binding.

Fig. 1.. Assay for specific binding of Ace1p to activated promoters of CUP1.

Ace1p-3xGFP (A) at the CUP1 array in YTK1498 or GFP-lacI (B) at pes4::LacO array in YTK1528 define the site for tracking of the individual molecules of Ace1p-HaloTag labeled with JF₆₄₆ at a low density of ≤ 5 particles per nucleus. Molecules of Ace1p-HaloTag (JF₆₄₆) frequently colocalize with CUP1 array and rarely colocalize with lacO array as demonstrated by single focal plane representative images. Gaussian filter was applied to Ace1p-HaloTag (JF₆₄₆) channel in Track Record software for better visualization of the particles. Here and in Figure 4B and 4C brightness and contrast were adjusted using linear LUT in Image J and cell contours were drawn manually in Adobe Illustrator. Scale bar = 2 μm. See also Movies S1 and S2. (C) Raw distributions of survival of Ace1p particles are shown with the exponential decay fits. For this Figure and for Figures 2 and 6A, pie charts represent the extracted distributions of diffusing (gray), specific (blue) and non-specific (orange) binding events with their average residence times; the table show the number of molecules / tracks observed for a particular sample. (D) The difference between residence times of Ace1p. ‘***’ - p<0.001 by z-test.

Fig. 2.. RSC affects transient binding of Ace1p at CUP1 promoter.

SMT of Ace1p at CUP1; charts, graphs and tables as described in the caption to Figure 1. Effect of Rsc2p depletion (A, YTK1649) or rsc2 knock out (B, YTK1526 vs wt YTK1498) on Ace1p binding at CUP1. (C) Compensation for depletion of Rsc2p by Rsc2p expressed from centromeric plasmid in YTK1582. (D) The average residence time of Ace1p at CUP1 from A-C. ‘n.s.’ - not significant 0.09<p<0.94, ‘*’ - p<0.05 by z-test.

Fig. 3.. RSC associates with active CUP1 locus and modulates its transcription and copper sensitivity of the cells.

(A) Binding of Rsc2p-AID*−9Myc to promoters of CUP1 upon activation with copper was assayed by ChIP. The effect of Rsc2p depletion on transcription of CUP1 quantified by RT-qPCR (B) and on copper sensitivity of the cells by colony growth assay (C). ‘*’ - p<0.05, ‘**’ - p<0.01, ‘***’ - p<0.001 by t-test.

Fig. 4.. RSC affects the “on” rate of Ace1p and the availability of RE at the CUP1 promoter.

(A) Diagram of the targeted integration of CUP1 reporter into CUP1 array (not to scale). The cassette contains a scrambled sequence derived from mammalian gene (scr. mam) and eight copies of the phage MS2 binding site, that may be transcribed in a single transcript, but cannot be translated. LEU2 is used as a selective marker for integration. Positions of the primers used to amplify the cassette are marked in magenta (T1010) and cyan (T1004). (B) Maximum Intensity projections of z-stack of a diploid cell containing the CUP1 reporter inserted into one of the two CUP1 arrays. TAMRA (red): Reporter mRNA stained by smFISH probes, Ace1p-3xGFP (green): CUP1 locus, DAPI (blue): nucleus. Overlays: CUP1 reporter mRNA colocalizes with only one of the two CUP1 transcription sites (arrowhead). Scale bar = 2 μm. (C) Population of cells with CUP1-reporter: transcribing (yellow circles), i.e. containing > 3 molecules of mature mRNA, and not transcribing (blue circles). Overlays of DAPI (green) and CUP1 reporter mRNA (red). Maximal Intensity Projections of the z-stacks of the images of the cells treated by 100 μM Cu²⁺ for CUP1 activation and 1 mM auxin for Rsc2 depletion and control cells of YTK1649 are presented. Original 15-bit images were scaled in Metamorph with linear LUT with the same range for brightness and contrast. Scalebar = 2 μm. (D) Counts of mature mRNA in control cells not activated by Cu²⁺ (upper panel, black bars) are fit to Poisson distribution with mean 0.46. The same Poisson distribution (mean = 0.46) can be used to fit the initial points of the distribution of mature mRNA in cells activated by Cu²⁺, both Rsc2⁺ and Rsc2p-depleted, if it is first scaled by a constant factor, α. (E) Histograms of mature mRNA per cell. The population of non-active cells (blue) was obtained by fitting the histogram cut off at 3 mRNA/cell to a scaled Poisson distribution with a fixed mean of 0.46 mRNA/cell estimated from the non-activated control. The scaling parameter in the fit is used to define the fraction of non-active cells, as represented in blue fonts.

Fig. 5.. RSC acts at pre-initiation stage.

(A) The production rate, t_prod for a single mRNA (pink line) is defined here as the time from initiation “I” through elongation “E” to release “R” of the mature mRNA. Mature mRNA then degrades with an average lifetime of k_deg⁻¹. (B) The gene switches between an ON state and an OFF state (black line). During a burst, transcription is initiated at random (green lines). The rate parameters characterize the frequency of bursts (k*_on and k_off), duration of a burst (k_off), and the amplitude of a burst (k_tx and k_off). (C) Histogram and bursting gene expression fits (red) of mature mRNA in Rsc2⁻, and Rsc2⁺. Fit lines were obtained by simulating the model with the average values of 1000 parameter sets that fit the data well. (D) Schematics of CUP1 burst dynamics in Rsc2⁺ and Rsc2⁻ cells based on parameters estimated by the gene busting model of transcription.

Fig. 6.. The role of RSC chromatin complex in nucleosomal mobility at CUP1.

(A) Single molecule tracking of Rsc2p-HaloTag (JF646) at CUP1 (YTK1635) and pes4::lacO (YTK1634). Charts and tables as described in the caption to Figure 1C. (B) Nucleosomal occupancy at CUP1. MNase-seq data for two biological replicate experiments. CUP1-2 - a single copy of CUP1 within the array. UASp,d - proximal and distal Upstream Activating Sequences, each containing two REs for Ace1p. Ovals - approximate positions of nucleosomes before activation. Treatment conditions: Rsc2⁻, Rsc2⁺, with or without Cu activation. (C) Proposed sequence of events at CUP1 promoter for an individual molecule of Ace1p: a molecule of RSC recruited to CUP1 mobilizes the −1 nucleosome, promoting binding of Ace1p; next, a bound molecule of Ace1p recruits the preinitiation complex (PIC); next, transcription starts, and PIC is disassembled; next, RSC moves the −1 nucleosome in opposite direction promoting eviction of Ace1p molecule.