Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells
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Evaluation Summary:
All three reviewers are in agreement that the study is of potential interest in the field of ER/PR signaling and endometrial cancer and that it contains significant amount of genomic data. However, functional data linking PAX2 to the PR/ER pathway are lacking, and the study is limited to a single model cell line and thus has a relatively narrow scope. There is also a concern that ChIP-seq data appear to be from a single, unreplicated experiment.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call ‘progestin control regions’ (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.
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Author Response:
Reviewer #1:
This manuscript explores the role of ER and PR in the endometrial cancer cell model called the Ishikawa cell line. The authors conduct a series of detailed experiments to assess the estrogen (E2) and progesterone (R5020) response in this model and show that E2 can promote cell growth which is subsequently inhibited by co-treatment with R5020. RNA-seq revealed E2 or R5020 gene targets with most differential genes being unique to the treatment condition. The functional role of PR was assessed and confirmed on a specific locus of interest and using a reporter assay. ChIP-seq was conducted revealing gained PR binding events following R5020 and some that were already present prior to treatment, as well as sites that were lost. Substantially more PR binding sites were observed in the T47 breast cancer model …
Author Response:
Reviewer #1:
This manuscript explores the role of ER and PR in the endometrial cancer cell model called the Ishikawa cell line. The authors conduct a series of detailed experiments to assess the estrogen (E2) and progesterone (R5020) response in this model and show that E2 can promote cell growth which is subsequently inhibited by co-treatment with R5020. RNA-seq revealed E2 or R5020 gene targets with most differential genes being unique to the treatment condition. The functional role of PR was assessed and confirmed on a specific locus of interest and using a reporter assay. ChIP-seq was conducted revealing gained PR binding events following R5020 and some that were already present prior to treatment, as well as sites that were lost. Substantially more PR binding sites were observed in the T47 breast cancer model and the authors mimic the elevated levels of PR, by expressing exogenous PR in Ishikawa cells and conducting a series of ChIP-seq experiments. Analysis of specific binding regions revealed the enrichment of motifs for the Pax family of transcription factors and the authors assess the hormonal regulation of PAX2 cellular expression. PAX2 ChIP-seq was conducted, revealing very few binding peaks and these were partially integrated with the PR and ER binding peaks. Finally, a Hi-C experiment was conducted, revealing that ER, PR and PAX2 binding occurs in genomic compartments and specific gene signatures were derived from this analysis.
This is a topical area and the work is of potential interest, but several key issues need to be addressed:
- The role for PAX2 (over other family members) is inferred by the enrichment for motifs specific to that family member, but the motif enrichments are not good as defining individual family members that share a common motif. What are the expression levels of the PAX family members in the Ishikawa cell line and in primary endometrial cancers, to support the role for PAX2 over other family members? This wouldn't require any experiments and could simply involve analysis of public expression datasets.
We agree with the reviewer that “the motif enrichments are not good as defining individual family members that share a common motif”. Besides choosing PAX2 because its motif was the most represented in PR and ERbs, we evaluated expression of PAX2 and other family members in publicly available normal and endometrial cancer samples. The expression levels of the PAX family members in Ishikawa cells and in primary endometrial cancer samples have been detailed in Figure 5, Figure 5-Figure Supplement 1, Figure 7 and Figure 7- Figure Supplement 1 (see new version of the manuscript). Although other PAX family members seem to be more expressed than PAX2 in tumors (like PAX8), many reports link the loss of PAX2 in endometrial tissue to bad prognosis of tumors (EIN) (see Sanderson et al, 2017 in revised manuscript). This was included in the discussion and now it has been included in the corresponding result argument connected to Figure 5 and Figure 7 of the revised manuscript.
- The authors conclude that PAX2 binding overlaps with PR in pre-treated cells, but data in Figure 5C and 5D could simply represent co-binding at open enhancers, which are notorious for recruiting many transcription factors that are expressed in that cell type. What is the overlap in peaks between PAX2 and PR/ER, ideally via a Venn diagram or some visual that allows for a comparison of the total number of peaks for each factor and the common ones? Is there a statistically enriched co-binding of PAX2 to PR/ER sites, at levels that are more than expected?
The fraction of overlap between sets of binding sites was studied in a pairwise fashion and is graphically represented in Figure 5C of the revised manuscript. Also, we performed a Fisher test to statistically evaluate the significance of the co-localization between PAXbs and hormone receptors which were included in Figure 5E and F. Results were included in text of revised manuscript (line 314).
- The link with PAX2 is potentially exciting but is not convincing in its current form. The only real evidence linking PAX2 to ER/PR is that PAX2 cellular location can be altered and there are some binding peaks where there is co-enrichment, but this would likely happen with any transcription factor expressed in that cell line model. What is currently missing (and essential), is some evidence providing a functional link between ER/PR and PAX2. Is PAX2 required for PR and/or ER function, either ER/PR binding or induction of target genes? If not, then the data on PAX2 is circumstantial and isn't really relevant to the transcriptional pathways regulated by PR or ER.
We completely agree with the reviewer. Functional data linking PAX2 to ER/PR signaling were studied using depletion of PAX2 with specific siRNA, showing clear effects on PR binding and hormone-regulated genes. These effects were observed in both E2 pretreated and non-pretreated cells, indicating that PAX2 is involved in many aspects of PR regulatory action and its interplay with ERalpha. Results on PR binding and gene expression with or without siRNA against PAX2 have been included in new Figure 5 (line 329 of revised manuscript). M&M and discussion were also revised accordingly.
- There is no explanation put forward to the 307 lost PR sites.
The sentence has been removed because we do not have replicates of 30min time point ChIPseq samples.
- The GEO dataset indicates that only one replicate was conducted for the ChIP-seq experiments. This does not meet the minimum ENCODE requirement and many of the differential peaks (i.e. the 307 lost peaks) are potentially false positives that result from having only one replicate.
We processed data from replicates and found similar results between them (see Figure 2 -Figure Supplement 2). While PRbs in E2-pretreated cells and ERbs were mostly reproduced (80% and 70%, respectively), PRbs in non- pretreated cells showed 40% of common peaks. This is mainly due to the fact that one of the replicates produced a higher number of peaks above threshold during peak calling, forcing a lower percentage of common peaks. However, we have demonstrated that almost all reported PRbs were independently reproduced by PR ChIP-seq data in other conditions, namely E2-pretreated PRbs and PRbs from FPR cells (Figure 2 and Figure 2-Figure Supplement 2). It is important to note that only PRbs from E2-pretreated cells and ERbs promoted the main conclusions of our manuscript. We will upload new data to GEO as soon as possible.
- The authors claim that the motif enrichment supports a conclusion where monomer PR could bind at 30 min and dimers at 60 min, but there is no direct evidence that this is the case. Unless the authors plan to pursue this functionally and can show dimeric vs monomeric binding, this statement should be removed, as it is not backed up by data and the presence of a half site vs a full palindromic motif does not provide evidence for the genuine mode of binding.
The statement has been removed.
- All the work is conducted in a single cell line model. I understand that there are few endometrial cancer cell line models and I also acknowledge that the authors have conducted a complicated series of genomic experiments and it would be unrealistic to repeat these in another model. However, the findings from this one model should reveal new insight that can be validated in either another model or in a cohort of clinical samples of the cancer types. But, in its current state, neither are done. The authors attempt to extract gene signatures from the genomic data to assess in patient cohorts, but the data (see my next comment) is not compelling or convincing and the only conclusion I can make, is that out of the hundreds of somatic mutations and hundreds of PgCR genes, only a handful of genes correlate with outcome. I suspect the same conclusion could be made with a random set of several hundred genes.
We reformulated the analysis of tumor samples to avoid biased conclusions. Results are part of the new version of Figure 7.
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Evaluation Summary:
All three reviewers are in agreement that the study is of potential interest in the field of ER/PR signaling and endometrial cancer and that it contains significant amount of genomic data. However, functional data linking PAX2 to the PR/ER pathway are lacking, and the study is limited to a single model cell line and thus has a relatively narrow scope. There is also a concern that ChIP-seq data appear to be from a single, unreplicated experiment.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript explores the role of ER and PR in the endometrial cancer cell model called the Ishikawa cell line. The authors conduct a series of detailed experiments to assess the estrogen (E2) and progesterone (R5020) response in this model and show that E2 can promote cell growth which is subsequently inhibited by co-treatment with R5020. RNA-seq revealed E2 or R5020 gene targets with most differential genes being unique to the treatment condition. The functional role of PR was assessed and confirmed on a specific locus of interest and using a reporter assay. ChIP-seq was conducted revealing gained PR binding events following R5020 and some that were already present prior to treatment, as well as sites that were lost. Substantially more PR binding sites were observed in the T47 breast cancer model and …
Reviewer #1 (Public Review):
This manuscript explores the role of ER and PR in the endometrial cancer cell model called the Ishikawa cell line. The authors conduct a series of detailed experiments to assess the estrogen (E2) and progesterone (R5020) response in this model and show that E2 can promote cell growth which is subsequently inhibited by co-treatment with R5020. RNA-seq revealed E2 or R5020 gene targets with most differential genes being unique to the treatment condition. The functional role of PR was assessed and confirmed on a specific locus of interest and using a reporter assay. ChIP-seq was conducted revealing gained PR binding events following R5020 and some that were already present prior to treatment, as well as sites that were lost. Substantially more PR binding sites were observed in the T47 breast cancer model and the authors mimic the elevated levels of PR, by expressing exogenous PR in Ishikawa cells and conducting a series of ChIP-seq experiments. Analysis of specific binding regions revealed the enrichment of motifs for the Pax family of transcription factors and the authors assess the hormonal regulation of PAX2 cellular expression. PAX2 ChIP-seq was conducted, revealing very few binding peaks and these were partially integrated with the PR and ER binding peaks. Finally, a Hi-C experiment was conducted, revealing that ER, PR and PAX2 binding occurs in genomic compartments and specific gene signatures were derived from this analysis.
This is a topical area and the work is of potential interest, but several key issues need to be addressed:
- The role for PAX2 (over other family members) is inferred by the enrichment for motifs specific to that family member, but the motif enrichments are not good as defining individual family members that share a common motif. What are the expression levels of the PAX family members in the Ishikawa cell line and in primary endometrial cancers, to support the role for PAX2 over other family members? This wouldn't require any experiments and could simply involve analysis of public expression datasets.
- The authors conclude that PAX2 binding overlaps with PR in pre-treated cells, but data in Figure 5C and 5D could simply represent co-binding at open enhancers, which are notorious for recruiting many transcription factors that are expressed in that cell type. What is the overlap in peaks between PAX2 and PR/ER, ideally via a Venn diagram or some visual that allows for a comparison of the total number of peaks for each factor and the common ones? Is there a statistically enriched co-binding of PAX2 to PR/ER sites, at levels that are more than expected?
- The link with PAX2 is potentially exciting but is not convincing in its current form. The only real evidence linking PAX2 to ER/PR is that PAX2 cellular location can be altered and there are some binding peaks where there is co-enrichment, but this would likely happen with any transcription factor expressed in that cell line model. What is currently missing (and essential), is some evidence providing a functional link between ER/PR and PAX2. Is PAX2 required for PR and/or ER function, either ER/PR binding or induction of target genes? If not, then the data on PAX2 is circumstantial and isn't really relevant to the transcriptional pathways regulated by PR or ER.
- There is no explanation put forward to the 307 lost PR sites.
- The GEO dataset indicates that only one replicate was conducted for the ChIP-seq experiments. This does not meet the minimum ENCODE requirement and many of the differential peaks (i.e. the 307 lost peaks) are potentially false positives that result from having only one replicate.
- The authors claim that the motif enrichment supports a conclusion where monomer PR could bind at 30 min and dimers at 60 min, but there is no direct evidence that this is the case. Unless the authors plan to pursue this functionally and can show dimeric vs monomeric binding, this statement should be removed, as it is not backed up by data and the presence of a half site vs a full palindromic motif does not provide evidence for the genuine mode of binding.
- All the work is conducted in a single cell line model. I understand that there are few endometrial cancer cell line models and I also acknowledge that the authors have conducted a complicated series of genomic experiments and it would be unrealistic to repeat these in another model. However, the findings from this one model should reveal new insight that can be validated in either another model or in a cohort of clinical samples of the cancer types. But, in its current state, neither are done. The authors attempt to extract gene signatures from the genomic data to assess in patient cohorts, but the data (see my next comment) is not compelling or convincing and the only conclusion I can make, is that out of the hundreds of somatic mutations and hundreds of PgCR genes, only a handful of genes correlate with outcome. I suspect the same conclusion could be made with a random set of several hundred genes.
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Reviewer #2 (Public Review):
Alejandro La Greca and co-workers have used the well-studied ER+/PR+ endometrial cancer cell line (Ishikawa cells) to model the genomic actions of ER and in particular PR in hormone-naïve and hormone-treated conditions using a variety of complementary techniques for probing global regulation of the genome (RNAseq, ChIPseq, ATACseq, Hi-C, CNV, and virtural 4C). They demonstrate that in contrast to the T47D breast cancer model, ER and PR mostly bind to cell-specific independent binding sites that are in close proximity to PAX2 binding sites. They define these regions as "progestin control regions" that exhibit an open chromatin state prior to hormone exposure. Hormone treatment results in the recruitment of PR and PAX2 as well as ER (at selected genes regulated by ER/PR/PAX2) to already formed loops in order …
Reviewer #2 (Public Review):
Alejandro La Greca and co-workers have used the well-studied ER+/PR+ endometrial cancer cell line (Ishikawa cells) to model the genomic actions of ER and in particular PR in hormone-naïve and hormone-treated conditions using a variety of complementary techniques for probing global regulation of the genome (RNAseq, ChIPseq, ATACseq, Hi-C, CNV, and virtural 4C). They demonstrate that in contrast to the T47D breast cancer model, ER and PR mostly bind to cell-specific independent binding sites that are in close proximity to PAX2 binding sites. They define these regions as "progestin control regions" that exhibit an open chromatin state prior to hormone exposure. Hormone treatment results in the recruitment of PR and PAX2 as well as ER (at selected genes regulated by ER/PR/PAX2) to already formed loops in order to initiate hormone-dependent transcription. The major strengths include that these studies reveal important differences between ER and PR crosstalk in uterine relative to breast cancer models and provide a deeper understanding of the mechanisms of PR-dependent regulation of the genome in well-studied endometrial cancer cells relative to well-studied breast cancer cell counterparts. Limitations of this work include the lack of additional ER+/PR+ models of endogenous steroid hormone receptor action to support a tissue-specific vs. cell line-specific role of PAX2 as a newly discovered partner with PR or ER/PR at hormone-regulated target genes. In addition, a requirement for PAX2 in PR-dependent gene regulation/gene selection has not been demonstrated.
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Reviewer #3 (Public Review):
The manuscript by La Greca et al explores the relationship between estradiol (E2) and progesterone (Pg) treatment with ER and PR binding and their crosstalk in the Ishikawa endometrial cancer cell line. They find that PR binding is enhanced upon treatment with the ER ligand, E2, and that overlap between ER and PR binding sites also corresponds with binding by the PAX2 transcription factor. The regions of PR and PAX2 binding, present in open chromatin regions inside TADs, are designated progestin control regions.
Although there is a high volume of high quality genomic data for a variety of hormone treatment conditions, there is an overall lack of functional relevance. The overlap and co-occupancy of ER and PR and PR and PAX2 is merely circumstantial. There are no experiments to show that PAX2 is important for …
Reviewer #3 (Public Review):
The manuscript by La Greca et al explores the relationship between estradiol (E2) and progesterone (Pg) treatment with ER and PR binding and their crosstalk in the Ishikawa endometrial cancer cell line. They find that PR binding is enhanced upon treatment with the ER ligand, E2, and that overlap between ER and PR binding sites also corresponds with binding by the PAX2 transcription factor. The regions of PR and PAX2 binding, present in open chromatin regions inside TADs, are designated progestin control regions.
Although there is a high volume of high quality genomic data for a variety of hormone treatment conditions, there is an overall lack of functional relevance. The overlap and co-occupancy of ER and PR and PR and PAX2 is merely circumstantial. There are no experiments to show that PAX2 is important for any of the transcriptional changes observed, or that loss of PAX2 would result in reduced PR binding or progestin-responsive genes. There is also very little explanation of what the function of PAX2 is in occupying adjacent chromatin sites to ER and PR. Additionally, many of the conclusions are based on small subsets of overlapping genes. There needs to be more exploration of what the consequences are of these overlaps in binding and adjacent binding sites. Finally, there is almost no discussion of what this genomic data means for endometrial cancer disease progression. It is primarily a description of ER, PR, and PAX2 binding in the Ishikawa cell line.
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