2018 Mar 2
Transcription-coupled Changes in Nuclear Mobility of Mammalian Cis-Regulatory Elements
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Transcription-coupled Changes in Nuclear Mobility of Mammalian Cis-Regulatory Elements
To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Conflict of interest statement
Competing interests: B.G., T.S., and J.W. have filed a U.S. provisional patent application relating to CARGO methodology. All other authors declare no competing interests.
Fig. 1.. CARGO-dCas9 imaging enables robust and noninvasive labeling of cis-regulatory elements in living cells.
A) CARGO assembly of a multiplexed gRNA array. Hybrid DNA oligonucleotides are synthesized with the first half of nth gRNA sequence, followed by the second half of the (n – 1)th gRNA separated by two BpiI restriction sites, with distinct sticky ends for each gRNA. Step 1: Synthetic DNA oligonucleotides are mixed and ligated with a permuted expression unit constant region (gRNA scaffold, Pol III termination signal, and human U6 promoter). Step 2: Resulting mini circles are cut with BpiI, exposing complementary sticky ends from different circles. Step 3: Digested products and destination vector are ligated to produce an array of gRNA expression units (shown in step 4) in a single-pot reaction. ( B) Representative examples of dCas9 imaging of genomic loci in mESCs. (Left) No gRNA control. (Middle) Single-gRNA–targeting telomere repeats. (Right) CARGO-array–targeting Fgf5 enhancer. Scale bars, 5 mm. ( C) Representative images showing colocalization of the Fgf5 enhancer CARGO dCas9-eGFP signal [as visualized by anti-eGFP (anti–enhanced GFP) immunofluorescence] with the DNA FISH signal (position of a BAC FISH probe is shown in fig. S3C). Scale bar, 2 mm. Colocalization was confirmed by Fisher’s exact test: P < 3 × 10 −28; odds ratio = 1.49 × 10 4 of nonrandom association between sparsely sampled dCas9 and DNA FISH image pixels. ( D and E) CARGO-dCas9 locus labeling efficiency (D) and signal-to-background ratio (E) in two clonal mESC lines (L1 and L2) bearing dCas9-GFP fusion and transfected with Fgf5 enhancer CARGO arrays. In (E), the bold line at the center of each box denotes the median value; top and bottom edges of the box denote the 25th and 75th percentiles, respectively.
Fig. 2.. Live-cell CARGO-dCas9 imaging and tracking of the
Fgf5 enhancer during differentiation of mESCs to mEpiLCs.
A) Live-cell two-dimensional (2D) tracking of the CARGO-dCas9–labeled Fgf5 enhancer in mESCs and mEpiLCs. (a and b) Movies of the cell nuclei are recorded as 2D projections of the 3D movement of the labeled loci. (c and d) Representative images of a single mESC (c) or mEpiLC (d) nucleus, overlaid with recorded trajectories color-coded by time (0 to 80 s). The bottom panels show zoomed-in views of the inset areas in the top images. Scale bars, 5 mm (top); 500 nm (bottom). ( B) Subdiffusive motion of the Fgf5 enhancer locus. tMSD for each tracked enhancer allele (colored curves) and eMSD (bold black curve, shaded area indicates ± SEM) as a function of the time interval (t) between observations. Ninety-one and 130 observed alleles are plotted for the mESC and mEpiLC state, respectively. The red dashed reference line has a slope of 0.5. ( C) Appearance of a fast-moving Fgf5 enhancer population in the mEpiLC state. Histograms of fitted apparent anomalous diffusion coefficients calculated from tMSD trajectories in mESCs or mEpiLCs, as indicated, are overlaid with fitted Gaussian mixture distribution curves in purple. Individual slow and fast components are plotted as blue and red curves, respectively. The inset bar plots indicate the number of recorded trajectories, together with the mixing proportion of slow and fast population components for each individual Gaussian. Bayesian information criterion (BIC) values for different component fittings are listed in table S1. The difference in distributions is supported by a two-sample Kolmogorov-Smirnov test.
Fig. 3.. Mobility of cis-regulatory elements changes with the transcriptional status of their associated genes.
A) Expression changes of genes whose cis-regulatory elements were analyzed by live-cell tracking. Ordinate: mean expression in mESC state; abscissa: mean expression in mEpiLC state, as measured by RNA sequencing. All genes are shown, with investigated genes highlighted in red. FPKM, fragments per kilobase of transcript per million mapped reads. ( B) Histograms of fitted apparent anomalous diffusion coefficients from tMSD of the indicated regulatory regions in mESCs (left panels) or mEpiLCs (right panels), overlaid with fitted Gaussian mixture distributions (purple) along with slow (blue) and fast (red) components. The inset bar plots indicate the number of recorded trajectories and the individual Gaussian mixing proportion of slow and fast population components. BIC values for different component fittings are listed in table S1. Differences in the distribution between the mESC and mEpiLC states are supported by a two-sample Kolmogorov-Smirnov test. ( C) Histogram of combined fitted apparent anomalous diffusion coefficients from tMSD of all loci in both mESC and mEpiLC states (n = 1271) overlaid with the fitted Gaussian mixture distribution (purple) along with slow (blue) and fast (red) components. Log-normal means and standard deviations of the slow and fast components (in μm 2 s −0.5) are denoted on the plot. ( D) Difference in fractions of fast populations between the mESC and mEpiLC states. Center bars indicate the median value; upper and lower bars indicate the 95% confidence interval of the estimates.
Fig. 4.. Inhibition of RNA polymerase II reverses activity-associated changes in enhancer mobility.
A) Live-cell locus tracking correlation with multiplexed single-molecule RNA FISH. Representative images of a cell with an inactive (top) or active (bottom) Fgf5 locus are shown. (Left) Snapshot of live-cell imaging in grayscale overlaid with fitted Fgf5 enhancer trajectories color-coded by frame number from 1 to 300 (corresponding to time 200 ms to 60 s). Scale bars, 5 mm. (Middle) Magnified view of the highlighted regions in the left panel. Scale bars, 500 nm. (Right) Multiplexed smFISH of Fgf5 mRNA from the same cells. Gray channel: DAPI (4′,6-diamidino-2-phenylindole) staining for cell nucleus; red channel: smFISH probe targeting the first exon of Fgf5 mRNA (the mCherry marker in the CARGO array is also visible); blue channel: smFISH probe targeting the first intron of Fgf5 mRNA. Colocalized intronic and exonic smFISH signals are highlighted by arrows. Scale bars, 5 mm. ( B) Mobility of the Fgf5 enhancer correlates with nascent transcription of the Fgf5 locus at the single-cell level. Cells were binned into three groups according to the transcription status of the Fgf5 locus, as measured by multiplexed smFISH and indicated at the bottom. Individual dots represent apparent anomalous diffusion coefficients extracted from the corresponding live-imaging tMSD data. Statistical significance is supported by a Kruskal-Wallis test. ( C) Increased mobility of the Fgf5 enhancer in mEpiLCs is reversed by Pol II inhibition. eMSD of the Fgf5 enhancer trajectories in mEpiLCs is shown before (blue circles) and after (red circles) treatment with DRB, flavopiridol, and triptolide, as indicated. ( D) Increased mobility of the Fgf5 enhancer in mEpiLCs is reversed by Pol II inhibition at the single-cell level. Anomalous diffusion coefficient of the Fgf5 enhancer is shown for the same cells before and after the corresponding drug treatment. Differences are supported by a paired Wilcoxon test, as indicated by P values in the plots. ( E) The stirring model provides an explanation for observed transcription-coupled changes in the mobility of cis-regulatory elements. The ground state (slow) is characterized by subdiffusive behavior with low apparent diffusivity governed by thermal forces. The activated (fast) state is characterized by an increased apparent diffusivity, which may be due to nonthermal agitation by transcribing RNA Pol II and/or its associated ATPases. Under the assumption that the radius of a local 3D chromosomal domain remains relatively invariant in the slow and fast states, elevated mobility of cis-regulatory elements would lead to decreased time to the first encounter between distally located enhancer and promoter regions, resulting in an increased enhancer-promoter contact frequency.
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