Ligand-dependent enhancer activation indirectly modulates non-target promoters in a chromatin domain
- Tata Institute of Fundamental Research Hyderabad, India
- National Centre for Biological Sciences, Tata Institute for Fundamental Research, India
- Sastra Deemed University, India
- School of Biotechnology, Amrita Vishwa Vidyapeetham, India
Figures
Figure 1 with 1 supplement
Estrogen receptor-alpha (ERα) binding and ligand-induced gene expression of TFF1 and TFF3 change over the course of estrogen signaling.
(A) Schematic depicting TFF1 locus, UCSC genome browser snapshots showing the binding of ERα, H3K27ac status, H3K4me3 signal, and GRO-seq signal for robustly estradiol (E2)-induced TFF1 locus. First, second, and third ERα ChIP-seq and GRO-seq tracks are from vehicle-treated, E2-1 hr and E2-3 hr in WT cells, respectively. (B) qRT-PCR showing the changes in expression of TFF1 and TFF3 genes during the E2 signaling time course. Error bars denote SEM from four biological replicates. Each dot represents a replicate. P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05.
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Figure 1—source data 1
qRT-PCR values for Figure 1B.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Experimental design.
(A) Schematic depicting the experimental design. Cells were cultured in complete media for 24 hr followed by stripping for 3 days. Finally, cells were induced for different durations with estradiol (E2)/Vehicle followed by different assays like single molecule RNA FISH (smFISH), 4C-seq, smFISH-IF, etc. (B) Zoomed in region around TFF3 gene, UCSC genome browser snapshots showing the binding of ERα and H3k4me3 signal. First, second, and third ERα ChIP-seq tracks are in vehicle-treated, E2-treated for 1 hr and E2-treated for 3 hr in WT cells, respectively. (C) UCSC genome browser snapshots showing GRO-seq signal for TFF3 gene. First, second, and third GRO-seq tracks are from vehicle-treated, E2-40m, and E2-160m in WT cells, respectively.
Figure 2 with 2 supplements
TFF1 and TFF3 expressions show opposite trends during the estradiol (E2) signaling time-course.
(A) 60 X Representative images from single molecule RNA FISH experiment showing transcripts for TFF1 and TFF3. The probe was designed against the unspliced RNA containing the intronic region. The scale bar is 5 microns. (B) The mean RNA numbers are depicted. These are counted using an in-house MATLAB code which uses the DAPI-stained nuclei as the mask to count the RNA present in the nucleus. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n=665, N=3). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (C) Scatter plots showing the distribution of InTFF1 and InTFF3 on a cell-by-cell basis (n=665, N=3). The absolute RNA numbers are combined from three different repeats. Density plots have been used to clearly visualize overlapping data points. (D) 60 X Representative images from single molecule RNA FISH experiment showing transcripts for InTFF1 and ExTFF1. Scale bar is 5 micrometers. (E) The mean RNA numbers for InTFF1 and ExTFF1 are depicted. Separate probes were used to target unspliced (InTFF1) and mature (ExTFF1) RNA. These are counted using an in-house MATLAB code which uses the DAPI-stained nuclei as the mask to count the intronic RNA present in the nucleus and a free-drawn region to designate the cell to count the exonic RNA present in the nucleus as well as the cytoplasm. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n>360, N=3). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (F) The mean RNA numbers for InTFF3 and ExTFF3 are depicted. Separate probes were used to target unspliced (InTFF3) and mature (ExTFF3) RNA. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n>210, N=3). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (G) Violin plots showing the ratio of intronic to exonic TFF1 counts are depicted. The graph shows the distribution of ratios combined from three different repeats (n>360, N=3). P-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (H) Violin plots showing the ratio of intronic to exonic TFF3 counts are depicted. The graph shows the distribution of ratios combined from three different repeats (n>210, N=3). P-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05.
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Figure 2—source data 1
Transcript counts from smFISH.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Dynamic induction and RNA localization of TFF1 and TFF3 transcription across cell populations using single molecule RNA (smRNA) FISH.
(A) Bar graph depicting the percentage of cells with 1, 2, 3, 4, or greater than four sites of transcription for TFF1 (left) is shown. The graph shows the mean of means from different repeats of the experiment, and error bars denote SEM (n>200, N=3). Only the cells with at least one allele firing were counted and cells with no alleles were not included in this. The graph on right shows the number of cells with zero or non-zero number of alleles firing. (B) Bar graph depicting the percentage of cells with 1, 2, 3, 4 or greater than four sites of transcription for TFF3 (left) is shown. The graph shows the mean of means from different repeats of the experiment, and error bars denote SEM (n>200, N=3). Only the cells with at least one allele firing were counted and cells with no alleles were not included in this. The graph in the middle shows the number of cells with 2, 3, 4, or greater than four sites of transcription for TFF3. The graph on the right shows the number of cells with zero or non-zero number of alleles firing. (C) Images from single molecule RNA FISH experiment showing transcripts for InTFF1 in cells induced for 1 hr with E2. The image shows that when a single allele of TFF1 is firing, the transcripts show a more spatially restricted localization. The scale bar is 5 microns. (D) Images from single molecule RNA FISH experiment showing transcripts for InTFF1 in uninduced cells. The image shows that when a single allele of TFF1 is firing and transcription is low, the transcripts show a more spatially restricted localization. The scale bar is 5 microns. (E) 60 X Representative images from a single molecule RNA FISH experiment showing transcripts for InTFF1 and ExTFF1 (top) and InTFF3 and ExTFF3 (bottom). The image shows that there is no intronic signal in the cytoplasm, while exonic signals can be found both in the nucleus and the cytoplasm. The scale bar is 5 microns. (F) 60 X Representative images from single molecule RNA FISH experiment showing transcripts for InTFF1 and ExTFF1. The image shows that all intronic signals are colocalized with exonic signals, but all exonic signals are expectedly not colocalized with intronic signals, representing more mature mRNA. The scale bar is 5 microns.
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Figure 2—figure supplement 1—source data 1
Percentage of cell from smFISH data.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
TFF1 and TFF3 expressions show opposite trends during the E2-signaling time-course.
(A) Violin plot showing the ratio of total intronic to absolute exonic TFF1 counts are depicted. Absolute exonic counts are calculated by subtracting total intronic transcripts from total exonic transcripts. The graph shows the distribution of ratios combined from three different repeats. Error bars denote SEM. P-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, and ns denotes p>0.05. (B) Violin plot showing the ratio of total intronic to absolute exonic TFF3 counts are depicted. Absolute exonic counts are calculated by subtracting total intronic transcripts from total exonic transcripts. The graph shows the distribution of ratios combined from three different repeats. Error bars denote SEM. P-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, and ns denotes p>0.05.
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Figure 2—figure supplement 2—source data 1
Ratio of intronic to exonic counts from smFISH data.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
Enhancer looping does not account for the differential expression of TFF1 and TFF3 genes.
(A) 4C-seq plot at TFF1 enhancer viewpoint, the interaction with the promoter is highlighted in yellow. The plot is overlaid with H3K27ac, ERα ChIP signal, and gene annotations. (B) Genome browser snapshot of TFF1 region depicting ERα binding in WT lines. The first, second, and third ERα ChIP-seq tracks are from WT cells that are vehicle-treated, E2-1 hr, and E2-3 hr, respectively. Blue highlighted regions represent the ΔTFF1e region. (C) The mean RNA numbers for InTFF1 in WT (unshaded) and ΔTFF1e (shaded) MCF7 cells are depicted. The mean of means are shown, and error bars denote SEM from three repeats (n>650, N=3 for each WT and delete line). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (D) The mean RNA numbers for InTFF3 in WT (unshaded) and ΔTFF1e (shaded) MCF7 cells are depicted. The mean of means are shown, and error bars denote SEM from three repeats (n>650, N=3 for each WT and delete line). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (E) The mean RNA numbers for InTFF1 and InTFF3 in ΔTFF1e MCF7 cells are depicted. The mean of means are shown, and error bars denote SEM from three repeats (n>880, N=3). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (F) Ratio of InTFF in WT MCF7 to InTFF in ΔTFF1e MCF7 are depicted. The ratio was obtained by dividing the absolute RNA counts of the WT line by delete lines performed on different days but in the same order (replicate one of WT divided by replicate one of ΔTFF1e). The mean of means are shown, and error bars denote SEM from three repeats (n>650, N=3 for each WT and delete line). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05.
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Figure 3—source data 1
Data for 4C (Figure 3A) and smFISH (Figure 3C–F).
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
TFF1 enhancer does not change target promoters during signaling time-course.
4C-seq plot at TFF1 enhancer viewpoint, the interaction with the promoter is highlighted in yellow. The plot is overlaid with H3K27ac, ERα ChIP signal, and gene annotations. There is no substantial interaction between the enhancer and TFF3 locus at any time point, while the interaction between the enhancer and TFF1 locus increases at 1hr and decreases at 3hr.
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Figure 3—figure supplement 1—source data 1
Data from 4C, replicate 2.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Nuclear levels of ERα dictate the extent of TFF1 and TFF3 expression.
(A) Representative images showing smFISH for InTFF1 and InTFF3 in combination with immunofluorescence for ERα (along with chromatin retention assay). The red circle denotes a cell with high ERα and low TFF1 and TFF3, the blue circle denotes a cell with medium ERα, high TFF1, and low TFF3 while the green circle denotes a cell with low ERα and high TFF3. The scale bar is 5 micrometres. (B) Histogram representing the distribution of ERα mean intensities in cells under uninduced, E2-1 hr, and E2-3 hr conditions (n=210). Intensities at 1 hr are the highest while they shift to the left at 3 hr and are lowest in uninduced cells. This is plotted from one experimental repeat out of three repeats as ERα intensities will vary from one immunofluorescence experiment to another. (C) Cumulative histogram representing the distribution of ERα mean intensities in cells under uninduced, E2-1 hr, and E2-3 hr conditions. P-values were calculated by the Mann-Whitney test and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. (D) ERα intensities were sorted into three categories namely low (intensities between 0–450 A.U.), mid (intensities between 450–1200 A.U.), and high (intensities between 1200–2100 A.U.). The mean and SEM of transcript count for InTFF1 in the three categories under uninduced, E2-1 hr, and E2-3 hr was plotted. Low and mid categories show the highest TFF1 mean. P-values were calculated by the Mann-Whitney test and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. This is plotted from one experimental repeat out of three repeats as ERα intensities will vary from one immunofluorescence experiment to another. (E) ERα intensities were sorted into three categories namely low (intensities between 0–450 A.U.), mid (intensities between 450–1200 A.U.), and high (intensities between 1200–2100 A.U.). The mean and SEM of transcript count for InTFF3 in the three categories under uninduced, E2-1 hr, and E2-3 hr was plotted. Low category shows the highest TFF3 mean. P-values were calculated by the Mann-Whitney test and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05. This is plotted from one experimental repeat out of three repeats as ERα intensities will vary from one immunofluorescence experiment to another. (F) 3D plot representing the distribution of ERα, InTFF1, and InTFF3 on a cell-by-cell basis shows that cells with lower levels of ERα show higher counts for InTFF3. This is plotted from one experimental repeat out of three repeats as ERα intensities will vary from one immunofluorescence experiment to another.
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Figure 4—source data 1
Data is from smFISH (TFF1 and TFF3) and Immunofluorscence (Estrogen receptor alpha).
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Levels of ERα in the nucleus dictate the extent of TFF1 and TFF3 inductions.
(A) Representative images from smFISH experiments showing InTFF1 and GAPDH (top panel) and InTFF1 and InTFF3 (bottom panel) in cells overexpressing ERα-GFP. Yellow circles show cells that are GFP positive and the transcripts associated with them. Note the visibly fewer TFF1, and TFF3 transcripts in the ERα-GFP-positive cells while GAPDH transcripts remain indistinguishable. This shows that ERα-GFP overexpression specifically affects the transcription of E2-regulated genes like TFF1 and TFF3 and not a housekeeping gene like GAPDH. This is further quantified in B and C. (B) Scatter plots showing the distribution of ERα-GFP intensities with GAPDH or InTFF1 transcript counts on a cell-by-cell basis from the experiment in the first row of A. Cells with high ERα-GFP expression can have high GAPDH expression but such cells necessarily have low TFF1 counts. (C) Scatter plots showing the distribution of ERα GFP intensities with InTFF1 or InTFF3 counts on a cell-by-cell basis from the experiment in the second row of A. As in B, high ERα-GFP expression leads to low expression of E2-regulated genes like TFF1 or TFF3. (D) Representative images showing smFISH for InTFF1 and InTFF3 in cells overexpressing EGFP-C1. Yellow circles show cells that are GFP positive and the transcripts associated with them. Just expressing EGFP in the same backbone as the ERα-GFP plasmid does not downregulate TFF1, and TFF3 expression; indicating that the downregulation effect is specific to ERα overexpression and not a generic effect of cell transfection. This is quantified in E. (E) Scatter plots showing the distribution of EGFP-C1 intensities with InTFF1 or InTFF3 transcript counts on a cell-by-cell basis. There is no obvious correlation between the levels of EGFP-C1 and TFF1 or TFF3, and a cell with high EGFP levels may well have large TFF1 or TFF3 transcript counts.
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Figure 4—figure supplement 1—source data 1
Data is from smFISH (TFF1, TFF3 and GAPDH) and ERa-GFP and GFP alone.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
1,6 HD exposure down-regulates TFF1 but supports TFF3 expression.
(A) Mean transcript counts for InTFF1 and InTFF3 in control, and 3% 1,6-Hexanediol treated cells post 30 min of E2- induction. The mean of means are shown, and the error bars denote SEM from three repeats (n>880, N=3). P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes P>0.05. (B) Boxplots showing DESeq2 normalized counts for low expressing, moderately expressing, and highly-expressing genes in the vehicle, E2-40m, and E2-160m, respectively (left). Boxplots showing DESeq2 normalized counts for genes near low expressing, moderately expressing, and highly expressing genes in the vehicle, E2-40m, and E2-160m, respectively (right). The p-values in the boxplots were calculated using the Wilcoxon rank-sum test. The boxplots depict the minimum, first quartile, median, third quartile, and maximum values, along with outliers. (C) Model depicting the signaling under uninduced, E2-1 hr, and E2-3 hr conditions – First, activation of target gene loci (like TFF1) occurs by ligand-dependent induction. During the active phase (1 hr), liganded ERα binds on enhancer and promoter. Together, these elements interact in 3D, manifesting as ERα punctae, which results in robust expression of target genes, and the sequestration of transcriptional machinery. At 3 hrs, as nuclear ERα decreases, the transcriptional machinery becomes available to other nearby promoters leading to increase in gene transcription at those loci.
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Figure 5—source data 1
smFISH counts for TFF1 and TFF3 upon E2 induction with and without 1,6HD.
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
LLPS perturbation down-regulates TFF1 but supports TFF3 expression.
Mean transcript counts for InTFF1 and InTFF3 in control, and 3% 1,6-hexanediol-treated cells in the absence of E2-induction show no difference in TFF1 counts on 1,6-hexanediol treatment. The bar denotes the mean of means from two repeats, and the error bars denote SEM. P-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p<0.001, ** denotes p<0.01, * denotes p<0.05, ns denotes p>0.05.
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Figure 5—figure supplement 1—source data 1
smFISH counts for TFF1 and TFF3 without E2 induction (with and without 1,6HD).
- https://cdn.elifesciences.org/articles/102417/elife-102417-fig5-figsupp1-data1-v1.xlsx
Additional files
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Supplementary file 1
Oligos and probe sequences.
(a) InTFF3 probe sequence. (b) ExTFF3 probe sequence. (c) InTFF1 probe sequence. (d) ExTFF1 probe sequence. (e) 4C Seq oligo sequences. (f) qPCR oligo sequences
- https://cdn.elifesciences.org/articles/102417/elife-102417-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/102417/elife-102417-mdarchecklist1-v1.docx
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Ligand-dependent enhancer activation indirectly modulates non-target promoters in a chromatin domain
eLife 13:RP102417.
https://doi.org/10.7554/eLife.102417.4
