Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells
- Biology and Experimental Medicine Institute, IBYME-CONICET, Argentina
- Institute of Nuclear Technologies for Health, INTECNUS-CONICET, Argentina
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
- Centre for Genomic Regulation (CRG), Barcelona Institute for Science and Technology, Spain
- Universitat Pompeu Fabra (UPF), Spain
- Bioscience Data Mining Group, Córdoba University, Argentina
Figures
Figure 1 with 3 supplements
R5020 inhibits E2-induced Ishikawa cell proliferation through an active PR that is capable of transactivating an exogenous MMTV promoter sequence and an endogenous enhancer sequence located 50 kb upstream of EGFR gene.
(A) Proliferation of Ishikawa cells either pretreated with E2 10 nM for 12 hr (preE2) or not (no preE2) and later treated with vehicle (OH), E2 (E2), R5020 (R5020), E2 combined with R5020 …
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Figure 1—source data 1
Proliferation assays in treated and untreated Ishikawa cells.
Measure represents number of cells x105 in the table.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig1-data1-v2.csv
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Figure 1—source data 2
Ishikawa mean nuclear signal intensity.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig1-data2-v2.csv
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Figure 1—source data 3
Ishikawa treated and untreated normalized expression dataset.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig1-data3-v2.txt
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Figure 1—source data 4
PR binding Ct values in EGFR enhancer sequence.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig1-data4-v2.csv
Figure 1—figure supplement 1
E2-dependent cell cycle progression and proliferation of Ishikawa cells is mediated by ERalpha.
R5020 antagonism on E2 is mediated by PR. (A) Cells were pretreated with ICI182780 (ICI ) and then treated with vehicle (OH) and E2 (E2) for 48 hr. Number of cells was determined and …
Figure 1—figure supplement 2
RNAseq results from hormone-treated Ishikawa cells.
(A) Overrepresented Biological Processes in RNAseq results from Ishikawa cells treated with R5020 and E2 for 12h analysed using DAVID web-based tool. Only the top 20 terms with p<0.05 were selected …
Figure 1—figure supplement 3
Ishikawa cells express six times less PR than T47D cells.
(A) Representative image of western blot assays using protein extracts from Ishikawa and T47D cells. Thirty µg (1) of T47D extracts were seeded followed by 10µg (1/3), 5µg (1/6), 3.3µg (1/9), and …
Figure 2 with 2 supplements
Estradiol induces R5020-dependent PR binding to specific regions in chromatin.
(A) Upper table shows total number of PRbs obtained by ChIPseq for untreated (0 min) and R5020-treated (5, 30 and 60 min) endometrial Ishikawa cells under three different conditions: non-pretreated …
Figure 2—figure supplement 1
Genome-wide analysis of PR and ERalpha binding in Ishikawa cells.
(A) ChIPseq results using anti-PR antibody in Ishikawa cells. Cells were treated or not (T0) for 5 min (R5), 30 min (R30) and 60 min (R60) with R5020 10 nM. The number of PRbs for T0, R30 and R60 is …
Figure 2—figure supplement 2
PR overexpression on Ishikawa cells.
(A) Validation of R5020-dependent PR binding in pretreated (+) or not (-) Ishikawa cells expressed as percentage of input (%input). Regions included are described in Figure 2—figure supplement 1B. …
Figure 3
A fraction of E2-induced PRbs localize on ERbs and contain half ERE motifs.
(A) Classification of steroid receptor binding relative to genomic features expressed as percentage (%) of peaks after 60 min of hormone treatment inside each feature. Legend at the top right corner …
Figure 4 with 1 supplement
Putative PAX2 binding sites are associated with PR and ER alpha binding and hormone-regulated genes in Ishikawa cells.
(A) Fold enrichment values (log2FE) of 1.395 known TF binding motifs on prePRbs and PRbs. Combined p-values for enrichment analyses are indicated through the color key displayed at the lower right …
Figure 4—figure supplement 1
PAX2 binding to chromatin is hormone dependent in Ishikawa cells while it is not localized to nuclei of T47D cells.
Scatter plots showing fold enrichment values (log2FE) of 1,395 known TF binding motifs on (A) PRbs in group I and group II from Figure 2—figure supplement 1G, (B) ERbs in group III and group IV from …
Figure 5 with 2 supplements
PAX2 co-localizes with PR and ERalpha in the nuclei of Ishikawa cells and it is positioned in the vicinity of PR and ER alpha binding sites.
(A) PAX2 expression in Non Tumor (normal endometrium) and two histologically distinct endometrial cancer samples (Dou et al., 2020). Data stored at the National Cancer Institute’s CPTAC program was …
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Figure 5—source data 1
PR recruitment measured by qPCR in PAX2-knocked down Ishikawa cells.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig5-data1-v2.csv
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Figure 5—source data 2
Gene expression measured by qPCR in PAX2-knocked down Ishikawa cells.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig5-data2-v2.csv
Figure 5—figure supplement 1
Expression analysis of PAX2 and other PAX family members in Ishikawa cells.
(A) Normalized expression of detected PAX family members in untreated and hormone-treated Ishikawa RNAseq samples. Expression is shown for two independent experimental replicates. (B) Representative …
Figure 5—figure supplement 2
Expression analysis of hormone regulated genes after PAX2 knock down.
(A) IGV-based visualization of treated Ishikawa ChIPseq data. PR, ERalpha, prePR, and PAX2 (R5020 and E2 sites) binding in regulatory regions of ALPP and TIPARP genes is shown in different tracks. (B…
Figure 6 with 1 supplement
Convergence of PR and PAX2 binding in TADs with regulated genes defines potential endometrial regulatory domains.
(A) Upper panel shows the contact matrices at a resolution of 20kb obtained by In Nucleo Hi-C in PGR and ALPP loci. Middle panel shows the spatial segregation of chromatin as open or closed …
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Figure 6—source data 1
TAD coordinates (hg38).
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig6-data1-v2.txt
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Figure 6—source data 2
PgCR coordinates (hg38).
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig6-data2-v2.txt
Figure 6—figure supplement 1
Chromosome compartments analysis and definition of progesterone control regions.
(A) Contact matrices of chr12 region at 100kb resolution obtained by In Nucleo Hi-C performed in two replicates of Ishikawa (Ishikawa_rep1 and Ishikawa_rep2) and T47D cells. (B) Hi-C datasets of …
Figure 7 with 1 supplement
Altered expression of PgCR-genes correlates with drivers of endometrial tumor progression.
(A) Scatter plot of PCA results showing scores of components 1 and 2 (PC1 and PC2) using transcriptomic data of endometrial cancer samples (protein coding genes) obtained from TCGA-UCEC database …
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Figure 7—source data 1
Intersection between tumor DEGs and PgCR-genes.
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig7-data1-v2.txt
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Figure 7—source data 2
Protein coding genes in TADs with PgCRs (PgCR-genes).
- https://cdn.elifesciences.org/articles/66034/elife-66034-fig7-data2-v2.txt
Figure 7—figure supplement 1
Exploratory analysis and processing of TCGA-UCEC endometrial cancer samples.
(A) Scatter plot of ESR1 and PGR expression levels for Stage I endometrial cancer samples. Orthogonal black dashed lines denote denote the quadrants for sample selection (colored quadrant). (B) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Edometrial adenocarcinoma (Epithelial) Ishikawa | Dr. Rochefort Unità Hormones and Cancer INSERM, France | Female | |
| Cell line (Homo sapiens) | FPR Ishikawa | This paper | Flag-tagged PR overexpression; Female | |
| Cell line (Homo sapiens) | Breast cancer (Epithelial) T47D | Dr. Beato (Center for Genomic Regulation) | Female | |
| Recombinant DNA reagent | p3xFLAG-CMV-14 (plasmid) | Dr. Beato (Center for Genomic Regulation) | Flag-tagged human PR | |
| Sequence-based reagent | Human PAX2 siRNA | Dharmacon | Pool of 4 on-target oligos | |
| Sequence-based reagent | Human control siRNA | QIAGEN 1027310 | Scramble non-target oligo | |
| Antibody | Anti-PR (H-190 Rabbit polyclonal) | Santa Cruz Bio. sc-7208 | RRID:AB_2164331 | ChIP:30µ l xIP Western (1:200) |
| Antibody | Anti-ERalpha (HC-20 Rabbit polyclonal) | Santa Cruz Bio. sc-543 | RRID:AB_631471 | Western (1:200) |
| Antibody | Anti-ERalpha (HC-20X Rabbit polyclonal) | Santa Cruz Bio. sc-543X | ChIP (25µ l xIP) | |
| Antibody | Anti-ERalpha (H-184X Rabbit polyclonal) | Santa Cruz Bio. sc-7204 | ChIP (25µ l xIP) | |
| Antibody | Anti-phosphoserine 294 PR (Rabbit polyclonal) | Cell Signaling 13736 | RRID:AB_2798307 | IF (1:100) |
| Antibody | Anti-PAX2 (Rabbit polyclonal) | BioLegend PRB-276P (Covance) | RRID:AB_291611 | ChIP:6µl xIP Western (1:200) |
| Antibody | Normal rabbit IgG | Santa Cruz Bio. sc-2027 | RRID:AB_737197 | ChIP (12µ l xIP) |
| Antibody | Anti-alphatubulin (Mouse monoclonal) | Merck T5168 (Sigma-Aldrich) | RRID:AB_477579 | Western (1:1000) |
