ERa binding and ligand-induced gene expression of TFF1 and TFF3 change over the course of estrogen signaling.

Schematic depicting TFF1 locus, UCSC genome browser snapshots showing the binding of ERα, H3K27ac status, H3K4me3 signal, and Gro-seq signal for robustly E2-induced TFF1 locus. First, second and third ERα ChIP-seq and Gro-seq tracks are from vehicle-treated, E2-1h and E2-3h 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.

TFF1 and TFF3 expressions show opposite trends during the E2 signaling time-course

A. 60X 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. 60X Representative images from single molecule RNA FISH experiment showing transcripts for InTFF1 and ExTFF1. Scale bar is 5 microns.

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.

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, E2-1h, and E2-3h, 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.

Nuclear levels of the transcription factor ERα dictate the extent of expression of TFF1 and TFF3 genes

A. Representative images showing smFISH for InTFF1 and InTFF3 in combination with immunofluorescence for ERα (along with chromatin retention assay). The blue circle denotes a cell with high ERα and low TFF1 and TFF3, the red circle denotes a cell with medium ERα, high TFF1, and low TFF3, while the cyan circle denotes a cell with low ERα and high TFF3. The scale bar is 5 microns.

B. Histogram representing the distribution of ERα mean intensities in cells under induced, E3-1hr, and E2-3hr conditions (n=210). Intensities at 1hr are the highest while they shift to the left at 3hr 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 induced, E3-1hr, and E2-3hr conditions (n=210). 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, E2-1hr, and E2-3hr were plotted (n=210). 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, and 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, E2-1hr, and E2-3hr were plotted. The 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 (n=210).

ERα mediated phase separation could govern the differential expression of TFF1 and TFF3 genes

A. Mean transcript counts for InTFF1 and InTFF3 in control, and 3% 1,6-Hexanediol treated cells post 30 minutes 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, E2-1hr, and E2-3h conditions-1. Activation of TFF1 locus by ligand induction. 2. During the active phase, liganded ERα binds on enhancer and promoter. Together these in 3D interact, manifesting as ERα puncta, which results in robust expression of target genes, 3. The phase-separated condensate leads to the sequestration of transcriptional machinery. 4. This favours intra-TAD enhancer: promoter interactions to facilitate robust gene transcription. Upon ERα degradation at 3h, these condensates disappear, and transcriptional machinery becomes available to other promoters leading to their somewhat increased gene transcription.

Experimental design

A. Schematic depicting the experimental design. Cells were cultured in complete media for 24h, followed by stripping for 3 days. Finally, cells were induced for different durations with E2/Vehicle followed by different assays like smFISH, 4C-seq, smFISH-IF, etc.

B. Zoomed in the region around the TFF3 gene, UCSC genome browser snapshots showing the binding of ERα and H3K4me3 signal. The first, second and third ERα ChIP-seq tracks are in vehicle-treated, E2 treated for 1h, and E2 treated for 3h in WT cells, respectively.

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.

TFF1 enhancer does not change target promoters during signalling 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.

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.

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.

InTFF3 probe sequence

ExTFF3 probe sequence

InTFF1 probe sequence

ExTFF1 probe sequence

4C Seq oligo sequences

qPCR oligo sequences