Assessment of the Epigenomic Landscape in Human Myometrium at Term Pregnancy

Reviewer #1 (Public Review):

Summary:

The use of a multi-omics approach to elucidate the regulatory mechanism underlying parturition and myometrial quiescence adds novelty to the study. The identification of myometrial cis-acting elements and their association with gene expression, particularly the regulation of the PLCL2 gene by PGR opens the door to further investigate the impact of PGR and other regulators.

Strengths:

(1) Multi-Omic Approach: The paper employs a comprehensive multi-omic approach, combining ChIP-Seq, RNA-Seq, and CRISPRa-based Perturb-Seq assays, which allow for a thorough investigation of the regulatory mechanisms underlying myometrial gene expression.

(2) Clinical Relevance: Investigating human myometrial specimens provides direct clinical relevance, as understanding the molecular mechanisms governing parturition and myometrial quiescence can have significant implications for the management of pregnancy-related disorders.

(3) Functional work: For functional screening, They have used CRISPRa-based screening of PLCL2 gene regulation using immortalized human cell-line hTERT-HM and T-hESC to add more dimension to the work which strengthens their finding of PGR-dependent regulation of the PLCL2 gene in the human myometrial cells.

Weaknesses:
(1) Variability in epigenomic mapping: The significant variations in the number and location of H3K27ac-positive intervals across different samples and studies suggest potential challenges in accurately mapping the myometrial epigenome. This variability may introduce uncertainty and complicate the interpretation of results.

(2) Sample specificity: The study focuses on term pregnant nonlabor myometrial specimens, limiting the generalizability of the findings to other stages of pregnancy or labor.

(3) Limited Understanding of Regulatory Mechanisms: While the study identifies potential regulatory programs within super-enhancers, the exact mechanisms by which these enhancers regulate gene expression and cellular functions in the myometrium remain unclear. Further mechanistic studies are needed to elucidate these processes.

(4) Discordant analysis: Why are regular enhancers being understood in terms of motif enrichment of transcription factors and super-enhancers in terms of pathways enriched for active genes? This needs a clear reason.

Reviewer #2 (Public Review):

Summary:

In "Assessment of the Epigenomic Landscape in Human Myometrium at Term Pregnancy" the authors generate a number of genome-wide data sets to investigate epigenomic and transcriptomic regulation of the myometrium at term pregnancy. These data provide a useful resource for further evaluation of gene regulatory mechanisms in the myometrium and include the first Hi-C data published for this tissue. There is a comprehensive comparison to previously published histone modification data and integration with RNA-seq to highlight potential enhancer-gene regulatory relationships. The authors further investigate putative enhancers upstream of the PLCL2 gene and identify a candidate region that may be regulated by the PGR (progesterone receptor) signaling.

Strengths:

The strengths of this study are in the multi-omics nature of the design as several genome-wide data sets are generated from the same patient samples. Extending this type of approach in the future to a larger number of samples will allow for additional investigation into gene regulation as the correlation between epigenomic features and gene expression across a larger number of samples can reveal regulatory relationships.

Weaknesses:

One of the most interesting aspects of this study is the generation of the first Hi-C data for the human pregnant myometrium, however, there is a minimal description in the results section of the Hi-C data analysis and the only data shown are the number of loops identified and one such loop that includes the PLCL2 promoter shown in Figure 3A. The manuscript would benefit from a more extensive analysis of the Hi-C data, for example, the analysis of TADs (topological associating domains) would be interesting to add and could be used to evaluate to what extent H3K27ac domains and putative regulated genes fall within the same TAD.

The authors present some convincing evidence on the transcriptional regulation of the PLCL2 gene using Perturb-Seq to identify putative upstream enhancer regions and PGR over-expression showing PGR can act as an activator. These two experiments on their own are interesting, however, they are not as mechanistically integrated as they could be to clarify the molecular mechanisms. Deletion of the putative enhancer upstream of PLCL2 followed by over-expression of PGR would clarify the mechanistic relationship between the proposed enhancer, PGR, and PLCL2 expression. Does PGR act through the proposed enhancer? In addition, reporter assays using this proposed enhancer region with and without increased expression of PGR and mutation of any PRE sequences would also provide mechanistic insight. Although CRISPRa and Perturb-Seq can be used to identify potential regulatory regions, the best approach to verify the requirement for a particular enhancer in regulating a specific gene is a deletion approach.

Reviewer #3 (Public Review):

In this manuscript, Wu et al. investigate active H3K27ac and H3K4me1 marks in term pregnant nonlabor myometrial biopsies, linking putative-enhancers and super-enhancers to gene expression levels. Through their findings, they reveal the PGR-dependent regulation of the PLCL2 gene in human myometrial cells via a cis-acting element located 35-kilobases upstream of the PLCL2 gene. By targeting this region using a CRISPR activation system, they were able to elevate the endogenous PLCL2 mRNA levels in immortalized human myometrial cells.

This research offers novel insights into the molecular mechanisms governing gene expression in myometrial tissues, advancing our understanding of pregnancy-related processes.

Major comments:

(1) A more comprehensive analysis of the epigenetic and transcriptomic data would have strengthened the paper, moving beyond basic association studies. Currently, it is challenging to assess the quality and significance of the data as much of the information is lacking.

(2) The rationale for and connections between experiments, as well as results, could be bolstered to underscore the significance of this research.

Strengths:

- The combination of ChIP-Seq, RNA-Seq, and CRISPRa Perturb-Seq approaches to investigate gene regulation and expression in myometrial cells.

- The use of CRISPR activation system to specifically target cis-acting elements.

Weaknesses:

- The manuscript would strongly benefit from a deeper analysis of the Omic datasets. Furthermore, expanding figures/graphs to effectively contextualize these datasets would be greatly beneficial and would add more value to this research. Currently, it is difficult for us to assess and appreciate the quality of these data sets across the manuscript, which is mostly correlative.

- Limited sample size, coupled with variability in results and overall lack of details, compromises the robustness of result interpretation.

- For most parts of the results section, a better description is needed, including rationale, approach, and presentation of data. As it stands, it is challenging to assess the quality of the data and appreciate the results.

- Additional efforts are needed to dissect the proposed regulatory mechanisms.

- While the discussion provided helpful context for understanding some of the experiments performed, it lacked interpretation of the results in relation to the existing literature.