Epigenetics: New Insights into Mammary Gland Biology
Round 1
Reviewer 1 Report
In this manuscript, the authors reviewed literature on the topic of epigenetics of mammary gland development and cancer. They covered DNA methylation and histone modifications and included detailed description of literature on various epigenetic regulators including polycomb proteins and non-coding RNAs. They discussed the roles of epigenetics in mammary cell fate specification and in various phases of mammary gland development including puberty, pregnancy, lactation, and involution. This is a timely subject as epigenetics is increasingly recognized to play critical roles in mammary gland development and tumorigenesis. However, in key sections such as sections 4 and 5, the authors provided publications in a somewhat catalog style without synthesizing them into a critical review of where the current state of the field is. Additional concerns are listed here:
“Virgin adult” and “mature adult” are used for two different ages groups here but these two terms sound very similar. Could the authors choose other terms that may better convey the age differences?
In line 65, the statement regarding non-coding RNA seems to be out of nowhere. Perhaps it should be deleted or moved to another place.
Substituting the word restricted for the word related might help in the statement “Epigenetic modifications are not related to a specific life stage of an organism but continue throughout the lifespan [8].”
Fig 2 is confusing and should be revised. It is unclear whether the components listed on the left regulate mammary gland function (such as development) or mammary cell epigenetics.
Please consider revising the sentence “Chromatin can be not transcribed and compact..” on page 106.
Revise “deletion of DNMT1 in mice leads to an important reduction in mammary stem/progenitor cells” as the word important reads awkward here.
References should be provided for the statement “A mouse model with mammary-specific TET2 deletion presents with impaired luminal lineage commitment resulting in enhanced ductal branching and an increased number and size of TEBs, as well as fibrosis and hyperplasic lesions in virgin heterozygous and knockout mice compared to wild type mice.”
In discussing JARID1B, papers that contradicted the H3K4me3 demethylation effects on deactivating gene transcription are presented without any interpretation.
The stem cell concept and stem cell assays seem to be confused in the following statement: Knockdown of JHDM1B leads to increased cellular proliferation in one cell line and increases colony-forming ability in both cell lines as well as greater stem cell potential, which explains the ability of knockdown cells to form mammospheres.
EZH2 germline knockout mice are embryonically lethal; therefore, “alternate models to conventional EZH2 knockout mice have therefore been used. A mouse model lacking EZH2 in mammary stem cells was generated.” While the first sentence is reasonable, mammary stem cell specific knockout is now trivial. No papers are not cited for this statement. On the other hand, here and in several other places in discussing difficulties in using germline knockout models, the authors seem to ignore tissue specific knockouts or mammary gland transplantation assays that are widely used to overcome germline lethality.
Fig 5 has no legend. It is unclear what these difficult colored circles mean.
Line 184: the phrase “the dry period” needs to be defined or replaced.
A typo: the word “are” needs to be deleted in “ductal elongation and branching are take place principally after puberty.”
Consider removing comma from “They can also be classified as guides, that regulate gene expression by recruiting enzymes that modify chromatin,” and from “They can be defined as scaffolds, that allow the formation of ribonucleoprotein complexes by recruiting proteins [32].”
Change “distinctiveorgan” to distinctive organ.
Author Response
Review 1
In this manuscript, the authors reviewed literature on the topic of epigenetics of mammary gland development and cancer. They covered DNA methylation and histone modifications and included detailed description of literature on various epigenetic regulators including polycomb proteins and non-coding RNAs. They discussed the roles of epigenetics in mammary cell fate specification and in various phases of mammary gland development including puberty, pregnancy, lactation, and involution. This is a timely subject as epigenetics is increasingly recognized to play critical roles in mammary gland development and tumorigenesis. However, in key sections such as sections 4 and 5, the authors provided publications in a somewhat catalog style without synthesizing them into a critical review of where the current state of the field is.
Answer: Syntheses have been added at the end of sections 4 and 5 (lines 225-233 and 307-312)
Additional concerns are listed here:
“Virgin adult” and “mature adult” are used for two different ages groups here but these two terms sound very similar. Could the authors choose other terms that may better convey the age differences?
Answer: The figures 1 and 3 have been modified.
In line 65, the statement regarding non-coding RNA seems to be out of nowhere. Perhaps it should be deleted or moved to another place.
Answer: The text has been modified, the sentence has been deleted and refences added line 133.
Substituting the word restricted for the word related might help in the statement “Epigenetic modifications are not related to a specific life stage of an organism but continue throughout the lifespan [8].”
Answer: The text has been modified (line 69).
Fig 2 is confusing and should be revised. It is unclear whether the components listed on the left regulate mammary gland function (such as development) or mammary cell epigenetics.
Answer: The text of the legend has been modified (lines 76-77).
Please consider revising the sentence “Chromatin can be not transcribed and compact..” on page 106.
Answer: The text has been modified (lines 115-116).
Revise “deletion of DNMT1 in mice leads to an important reduction in mammary stem/progenitor cells” as the word important reads awkward here.
Answer: The text has been modified (line 174).
References should be provided for the statement “A mouse model with mammary-specific TET2 deletion presents with impaired luminal lineage commitment resulting in enhanced ductal branching and an increased number and size of TEBs, as well as fibrosis and hyperplasic lesions in virgin heterozygous and knockout mice compared to wild type mice.”
Answer: Reference has been added (line 179, ref 47)
In discussing JARID1B, papers that contradicted the H3K4me3 demethylation effects on deactivating gene transcription are presented without any interpretation.
Answer: The text has been modified and data discussed (lines 256-264).
The stem cell concept and stem cell assays seem to be confused in the following statement: Knockdown of JHDM1B leads to increased cellular proliferation in one cell line and increases colony-forming ability in both cell lines as well as greater stem cell potential, which explains the ability of knockdown cells to form mammospheres.
Answer: The text has been modified and clarifications have been reported (line 289-300).
EZH2 germline knockout mice are embryonically lethal; therefore, “alternate models to conventional EZH2 knockout mice have therefore been used. A mouse model lacking EZH2 in mammary stem cells was generated.” While the first sentence is reasonable, mammary stem cell specific knockout is now trivial. No papers are not cited for this statement. On the other hand, here and in several other places in discussing difficulties in using germline knockout models, the authors seem to ignore tissue specific knockouts or mammary gland transplantation assays that are widely used to overcome germline lethality.
Answer: Additional clarifications have been reported in the text (lines 338-342).
Fig 5 has no legend. It is unclear what these difficult colored circles mean.
Answer: Figure 5 has been modified.
Line 184: the phrase “the dry period” needs to be defined or replaced.
Answer: “the dry period” expression has been explained (line 196).
A typo: the word “are” needs to be deleted in “ductal elongation and branching are take place principally after puberty.”
Answer: The text has been modified (line 46).
Consider removing comma from “They can also be classified as guides, that regulate gene expression by recruiting enzymes that modify chromatin,” and from “They can be defined as scaffolds, that allow the formation of ribonucleoprotein complexes by recruiting proteins [32].”
Answer: The text has been modified (lines 138 and 139).
Change “distinctiveorgan” to distinctive organ.
Answer: The text has been modified (line 148).
Author Response File: 
Author Response.docx
Reviewer 2 Report
Ivanova et al. have summarized the available data on main molecular mechanisms involved in epigenetics in normal mammary gland biology. This is well written manuscript.
Comments:
- It might be easier for general audiences to follow the manuscript if it begins with four main epigenetic mechanisms a (section 3, Fig2) then introduce mammary gland (section 2, Fig1) and dive into each epigenetic modification according to developmental stage.
- If the journal allows more figures or tables, please add more for Histone modifications, Polycomb proteins role, and Roles of miRNAs for cell fate, linage and differentiation.
- Figure 5: please explain the color codes in the legend.
Thank you.
Author Response
Review 2
Ivanova et al. have summarized the available data on main molecular mechanisms involved in epigenetics in normal mammary gland biology. This is well written manuscript.
Comments:
- It might be easier for general audiences to follow the manuscript if it begins with four main epigenetic mechanisms a (section 3, Fig2) then introduce mammary gland (section 2, Fig1) and dive into each epigenetic modification according to developmental stage.
Answer: The propositions of the reviewer 2 have been discussed by the authors before the redaction of the review. The two options were possible and finally we have chosen to describe the mammary biology first to allow the reader to have all information concerning the organ for which epigenetic regulations are described.
Concerning the description of epigenetic modifications according to developmental stages, most of papers described data in several stages and as the mammary biology depend of the continuum of these stages, the description stage by stage is difficult and lot of papers will be described several times.
- If the journal allows more figures or tables, please add more for Histone modifications, Polycomb proteins role, and Roles of miRNAs for cell fate, linage and differentiation.
Answer: The implications of epigenetic regulators in cell proliferation, viability or differentiation are presented in figure 4. As level of information provides by each paper is different, it is not possible to propose summary figure with more details.
- Figure 5: please explain the color codes in the legend.
Answer: Figure 5 has been modified.
Author Response File: Author Response.docx
Reviewer 3 Report
This review offers a nice overview of the current knowledge on the role of epigenetic mechanisms of mammary gland development. The article is well written with small grammatical errors throughout. The mentioned studies are well described, and the tables and figures are informative. I have a couple of minor comments related to some statements, that are quite bold in the present form, and could mislead readers.
The chapter 3: Epigenetic modification The sentence in line 67 “Epigenetic modification are not related to a specific life stage” could be misleading. While I agree with the point that epigenetic marks are dynamic and responsive to environmental cues throughout life. However, there are several developmental periods (prenatal, early postnatal, transition to puberty) which are more vulnerable periods for epigenetic disruption and authors should briefly discus that in a context of mammary gland development.
3.1. DNA methylation It would be useful if authors mention the relationship between DNA methylation and the regulation of gene expression in a context of non-CpG sites. Also, authors should mention that many other genomic regions (for example gene bodies) might be subjected to DNA methylation resulting in the repression or activation of genes, depending on the context.
3.4 Noncoding RNAs Line 139 “The mammary gland is distinctive organ in its capability to undergo…” This statement is quite bold, since uterus also undergoes cellular remodeling which combines proliferation and apoptosis during each menstrual cycle.
Author Response
Review 3
This review offers a nice overview of the current knowledge on the role of epigenetic mechanisms of mammary gland development. The article is well written with small grammatical errors throughout. The mentioned studies are well described, and the tables and figures are informative. I have a couple of minor comments related to some statements, that are quite bold in the present form, and could mislead readers.
The chapter 3: Epigenetic modification The sentence in line 67 “Epigenetic modification are not related to a specific life stage” could be misleading. While I agree with the point that epigenetic marks are dynamic and responsive to environmental cues throughout life. However, there are several developmental periods (prenatal, early postnatal, transition to puberty) which are more vulnerable periods for epigenetic disruption and authors should briefly discus that in a context of mammary gland development.
Answer: The text has been modified and this point has been discussed (lines 70-73).
3.1. DNA methylation It would be useful if authors mention the relationship between DNA methylation and the regulation of gene expression in a context of non-CpG sites. Also, authors should mention that many other genomic regions (for example gene bodies) might be subjected to DNA methylation resulting in the repression or activation of genes, depending on the context.
Answer: The text has been modified and this point has been added (lines 94-97).
3.4 Noncoding RNAs Line 139 “The mammary gland is distinctive organ in its capability to undergo…” This statement is quite bold, since uterus also undergoes cellular remodeling which combines proliferation and apoptosis during each menstrual cycle.
Answer: The text has been modified (line 148).
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors have largely addresses my concerns and the manuscript has significantly improved.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
