Reviewer's report
Title:Mapping of HNF4alpha target genes in intestinal epithelial cells unravels a
gene regulatory network involving Cdx-2.
Version: 1Date:8 March 2009
Reviewer: Jean-Noel FREUND
Reviewer's report:
In this study, Boyd and coworkers have combined a transcriptional microarray
approach and a ChIP-on-chip approach to address the regulatory network driven
by the transcription factor HNF4g in the human intestinal cell line Caco-2. In the
first part of the study, the authors present a computer-based analysis of the data
and in the second part they focus on three promoters, namely the promoters of
the homeobox gene CDX2, of the disaccharidase Trehalase, and of a component
of tight junctions, cingulin. This work is very interesting as it provides a lot of new
information about the complexity of the network governed by HNF4g. However,
several issues have to be addressed to improve the manuscript.
Major issues
- A major point is that the authors have correlated the presence of HNF4g
binding sites in the promoters with changes in gene expression levels during cell
differentiation instead of changes in gene expression levels related to HNF4g
changes (ie after knockdown by siRNA or by overexpression using expression
plasmids). This should be discussed better than only mentioning the difficulties to
knockdown HNF4g by siRNA or shRNA.
- Table I is rather obscure. In particular, it should be better explained how the
numbers under parentheses are calculated. Second, how were chosen the 35
genes shown in this Table? It could be interesting to rank the list of genes either
with respect to the ChIP data or to the expression levels, and also provide a list
of genes positive in ChIP experiments but negative in expression levels. What is
the position of the 3 promoters analyzed in detail later in this ranking?
- In the Fig 3, 4 and 5, the luciferase data are missing in the present version of
the manuscript (supposed to be sections E of these figures).
- For the luciferase assays, the authors used Cos7 cells, which are far from being
a model of cell differentiation in vitro. Although these cells have commonly been
used to study the transcriptional activity of HNF4g, data have also to be provided
in intestinal cells. For instance, an intestinal cell line responsive to HNF4g has
actually been used in luciferase assays to show the activation of CDX2
expression by HNF4g (Ref 58).
- An important reference is missing to further interpret the data on HNF4g,
cingulin and cell-cell interaction: Peignon et al J Biol Chem 2006;281:3560-8
Minor issues
- It is claimed in p11 that CDX2 was not known to be a downstream target of
HNF4g, but in fact it was, as mentioned in the discussion (Ref 58).
- In Fig 3B, the CDX2 promoter region with highest ChIP (around -300) has a low
level of acetylated histones, which is not what is reported in the text.
- The title of the manuscript does not accurately convey what has been found.
First, one would expect from the title that the authors have compared the gene
networks driven by HNF4g and CDX2. Second, the authors point to CDX2 in the
title, but in the last figure, they involve not only CDX2 but also HNF1.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:Mapping of HNF4alpha target genes in intestinal epithelial cells unravels a
gene regulatory network involving Cdx-2.
Version: 1Date:8 March 2009
Reviewer: Jean-Noel FREUND
Reviewer's report:
In this study, Boyd and coworkers have combined a transcriptional microarray
approach and a ChIP-on-chip approach to address the regulatory network driven
by the transcription factor HNF4g in the human intestinal cell line Caco-2. In the
first part of the study, the authors present a computer-based analysis of the data
and in the second part they focus on three promoters, namely the promoters of
the homeobox gene CDX2, of the disaccharidase Trehalase, and of a component
of tight junctions, cingulin. This work is very interesting as it provides a lot of new
information about the complexity of the network governed by HNF4g. However,
several issues have to be addressed to improve the manuscript.
Major issues
- A major point is that the authors have correlated the presence of HNF4g
binding sites in the promoters with changes in gene expression levels during cell
differentiation instead of changes in gene expression levels related to HNF4g
changes (ie after knockdown by siRNA or by overexpression using expression
plasmids). This should be discussed better than only mentioning the difficulties to
knockdown HNF4g by siRNA or shRNA.
- Table I is rather obscure. In particular, it should be better explained how the
numbers under parentheses are calculated. Second, how were chosen the 35
genes shown in this Table? It could be interesting to rank the list of genes either
with respect to the ChIP data or to the expression levels, and also provide a list
of genes positive in ChIP experiments but negative in expression levels. What is
the position of the 3 promoters analyzed in detail later in this ranking?
- In the Fig 3, 4 and 5, the luciferase data are missing in the present version of
the manuscript (supposed to be sections E of these figures).
- For the luciferase assays, the authors used Cos7 cells, which are far from being
a model of cell differentiation in vitro. Although these cells have commonly been
used to study the transcriptional activity of HNF4g, data have also to be provided
in intestinal cells. For instance, an intestinal cell line responsive to HNF4g has
actually been used in luciferase assays to show the activation of CDX2
expression by HNF4g (Ref 58).
- An important reference is missing to further interpret the data on HNF4g,
cingulin and cell-cell interaction: Peignon et al J Biol Chem 2006;281:3560-8
Minor issues
- It is claimed in p11 that CDX2 was not known to be a downstream target of
HNF4g, but in fact it was, as mentioned in the discussion (Ref 58).
- In Fig 3B, the CDX2 promoter region with highest ChIP (around -300) has a low
level of acetylated histones, which is not what is reported in the text.
- The title of the manuscript does not accurately convey what has been found.
First, one would expect from the title that the authors have compared the gene
networks driven by HNF4g and CDX2. Second, the authors point to CDX2 in the
title, but in the last figure, they involve not only CDX2 but also HNF1.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Mapping of HNF4alpha target genes in intestinal epithelial cells unravels a
gene regulatory network involving Cdx-2.
Version: 1Date:8 March 2009
Reviewer: Jean-Noel FREUND
Reviewer's report:
In this study, Boyd and coworkers have combined a transcriptional microarray
approach and a ChIP-on-chip approach to address the regulatory network driven
by the transcription factor HNF4g in the human intestinal cell line Caco-2. In the
first part of the study, the authors present a computer-based analysis of the data
and in the second part they focus on three promoters, namely the promoters of
the homeobox gene CDX2, of the disaccharidase Trehalase, and of a component
of tight junctions, cingulin. This work is very interesting as it provides a lot of new
information about the complexity of the network governed by HNF4g. However,
several issues have to be addressed to improve the manuscript.
Major issues
- A major point is that the authors have correlated the presence of HNF4g
binding sites in the promoters with changes in gene expression levels during cell
differentiation instead of changes in gene expression levels related to HNF4g
changes (ie after knockdown by siRNA or by overexpression using expression
plasmids). This should be discussed better than only mentioning the difficulties to
knockdown HNF4g by siRNA or shRNA.
- Table I is rather obscure. In particular, it should be better explained how the
numbers under parentheses are calculated. Second, how were chosen the 35
genes shown in this Table? It could be interesting to rank the list of genes either
with respect to the ChIP data or to the expression levels, and also provide a list
of genes positive in ChIP experiments but negative in expression levels. What is
the position of the 3 promoters analyzed in detail later in this ranking?
- In the Fig 3, 4 and 5, the luciferase data are missing in the present version of
the manuscript (supposed to be sections E of these figures).
- For the luciferase assays, the authors used Cos7 cells, which are far from being
a model of cell differentiation in vitro. Although these cells have commonly been
used to study the transcriptional activity of HNF4g, data have also to be provided
in intestinal cells. For instance, an intestinal cell line responsive to HNF4g has
actually been used in luciferase assays to show the activation of CDX2
expression by HNF4g (Ref 58).
- An important reference is missing to further interpret the data on HNF4g,
cingulin and cell-cell interaction: Peignon et al J Biol Chem 2006;281:3560-8
Minor issues
- It is claimed in p11 that CDX2 was not known to be a downstream target of
HNF4g, but in fact it was, as mentioned in the discussion (Ref 58).
- In Fig 3B, the CDX2 promoter region with highest ChIP (around -300) has a low
level of acetylated histones, which is not what is reported in the text.
- The title of the manuscript does not accurately convey what has been found.
First, one would expect from the title that the authors have compared the gene
networks driven by HNF4g and CDX2. Second, the authors point to CDX2 in the
title, but in the last figure, they involve not only CDX2 but also HNF1.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
assistant