TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells

Abstract

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

Figure 1.

Schematic illustration of the TriTag strategy. Schematic representation of the TriTag imaging system for visualizing DNA, nascent RNA and protein in a single living cell. This system combines three imaging capabilities, including DNA labeling via the CRISPR–Cas9 system, RNA tracking by the MS2-MCP approach, and protein imaging using fluorescent proteins. As an example, a hybrid DNA sequence of CRISPR-Tag and MS2 aptamer is embedded in the intron of blue fluorescent protein (BFP) coding sequence. Transcription is a process where the DNA of a gene is copied to make nascent messenger RNA (mRNA). Nascent mRNA is then spliced to produce mature mRNA, which is later used as instructions to make proteins. CRISPR-Tag is recognized by dCas9-GFP at the DNA level, whereas nascent RNA is bound by stdMCP-tdTomato at the RNA level. Then, the fluorescent protein is expressed. Finally, DNA, nascent mRNA, and protein can be visualized in the same cell by fluorescent imaging.

Figure 2.

Tricolor visualization of DNA, RNA, and protein in living cells using TriTag. (A) Schematic of TriTag design. A hybrid of MS2 loops and CRISPR-Tag was first generated and then inserted in the intron of blue fluorescent protein (BFP). (B) Representative images to show simultaneous visualization of DNA, RNA and protein of H2B (top and middle) or LMNA (bottom) genes in HeLa cells. All images are from single focal plane. Scale bars: 5 μm. (C) Line scan of the relative fluorescence of the signal indicated by the dotted lines in (C). (D) Statistics of signal-to-noise ratio (SNR) to report the labeling of nascent transcripts at H2B and LMNA loci, n ≥ 90 genomic loci. Each dot represents a genomic locus. Green line denotes means ± SEM. (E) Statistics of signal-to-noise ratio to demonstrate DNA labeling efficiency at H2B and LMNA loci with or without transcription respectively, n ≥ 97 genomic loci. Each dot represents a genomic locus. Green line denotes means ± SEM. (F) Bar graph showing the size of dCas9-GFP_14X spots representing H2B or LMNA loci, with and without transcription, respectively. n ≥ 50 genomic loci. Error bar represents means ± SEM. ***P < 0.0001. (G) FACS analysis of H2B-BFP or BFP-LMNA expression in HeLa cells. mCherry serves as an irrelevant channel. The distribution of dots along the x-axis indicates BFP signal intensity. Each dot represents a single cell.

Figure 3.

TriTag enables real-time imaging of RNA synthesis and protein expression. (A, B) Representative traces (red) of nascent transcripts enriched at H2B (A) or LMNA (B) loci compared to that of background signal (gray), illustrating their bursting kinetics. Some snapshots at indicated time points are taken from the cell that was used to generate the corresponding traces. See Movie S2 for dynamics of H2B transcriptional bursting. (C) Bar graph showing the average value of total duration time of all bursts occurred within one hour at H2B or LMNA loci, respectively, n ≥ 50 cells. Error bars represent means ± SEM. (D) Representative images illustrating the simultaneous labeling of BFP protein and its nascent RNAs. The expression of BFP was driven by two promoters, SFFV (top) and CMV (bottom). All images are maximum intensity projections from z stacks. Scale bars: 5 μm. (E) Representative traces (red) demonstrating the transcriptional bursting patterns of SFFV and CMV, respectively. Gray traces indicate the intensity of the background signal. (F) Histograms of burst durations (n = 157 bursts) and pause durations (n = 122 pauses) of SFFV promoter. (G) Histograms of burst durations (n = 157 bursts) and pause durations (n = 123 pauses) of CMV promoter. (H) Scatter plots of BFP expression level and the amount of total nascent transcripts in the same cell. Green line denotes the linear fit. R² represents the coefficient of determination. Each dot represents a single cell, n ≥ 47 cells.

Figure 4.

TriTag monitors allele-specific transcriptional bursting across the cell cycle. (A) Fluorescent images of a cell showing DNA labeling of two LMNA alleles labeled by dCas9-GFP_14X and their transcriptional kinetics indicated by stdMCP-tdTomato at multiple time points. See Movie S3 for dynamics. (B, C) Line scan of intensity profiles illustrating dynamic labeling of DNA (green) and nascent RNAs (red) of locus 1 (B) and locus 2 (C) indicated in (A). (D) Representative images revealing three distinct transcriptional states of sister chromatid pairs identified by co-labeling of dCas9-GFP_14X and stdMCPtdTomato. (E) Line scan of intensity profiles showing dynamic labeling of DNA and nascent RNAs at corresponding sister chromatids pointed by arrows in (D). (F) Sister chromatids of the same allele have correlated transcription activity. Intensities of both transcription sites were plotted, with each dot representing a different pair of sister chromatids, n = 240 pairs. Green line denotes the linear fit. R² represents the coefficient of determination. (G) Bar graph showing transcription states of two LMNA alleles in the same cell (case in A, n = 284 cells) or pairs of sister chromatids (case in D, n = 229 cells). (H) Snapshots of the transcription activity of LMNA gene in HeLa cells at indicated time points through mitosis. See Movie S4 and S5 for dynamics. (I) Transcriptional activity of the mother cell and its daughter cells at LMNA loci was quantified to generate the scatter plot. Each dot represents a pair of the mother cell and one of its daughter cells, n = 90 pairs. Green line denotes the linear fit. R² represents the coefficient of determination. (J) Bar graph showing quantifications of transcriptional activity in daughter cells derived from three groups of mother cells with no, low and high transcription of LMNA gene, respectively. n ≥ 52 cells. All scale bars: 5 μm.

Figure 5.

Transcription-coupled chromatin dynamics revealed by the TriTag system. (A) Colocalization of dCas9-GFP_14X and stdMCP-tdTomato demonstrates the transcription status of LMNA alleles. BFP-LMNA was captured to highlight the nuclear membrane. Scale bar: 5 μm. (B) Movement trajectories of dCas9-GFP_14X spots representing the two indicated loci in (A). The trajectory lengths are 400 frames with 0.25 s per frame. Scale bars: 100 nm. (C) The averaged mean square displacement (MSD) curves of LMNA loci with (TF on) or without (TF off) transcription. The data are displayed as means ± SEM for 44 pairs of alleles. Each pair was from the same cell, with one active and the other one silent. The shaded areas represent the SEM. (D, E) Quantifications of the confinement area (D) and the microscopic diffusion coefficient (E) estimated from the MSD curves in (C). Red line at the center of each box denotes the median value, top and bottom edges indicate the 25th and 75th percentiles, respectively. (F) Live-cell imaging snapshots of a cell showing chromatin dynamics and transcriptional activation of the HSPA1A gene under the stress condition (42°C, 0.03% CO₂). See Movie S6 for dynamics. Scale bar: 5 μm. (G) Quantifications of the transcription activity (top) defined by the total intensity of stdMCP-tdTomato spots, and chromatin dynamics (bottom) by measuring the size of dCas9-GFP_14X spots, respectively. Three representative cells were measured, including the one from (F). (H) Live-cell imaging snapshots of a cell illustrating chromatin dynamics and transcription activation with the addition of triptolide under the same stress condition as (F). See Movie S7 for dynamics. Scale bar: 5 μm. (I) Quantifications were performed as those in (G) for the condition in (H). Three representative cells were analyzed, including the one from (H).