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 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 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.

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 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, E2-1hr, 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 uninduced, E2-1hr, and E2-3hr 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, E2-1hr, and E2-3hr 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, E2-1hr, and E2-3hr 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.

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 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 E2-3hr 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.