Reviewer's report
Title:Mapping of HNF4alpha target genes in intestinal epithelial cells unravels a
gene regulatory network involving Cdx-2.
Version: 1Date:27 March 2009
Reviewer: Oliver Burk
Reviewer's report:
This manuscripts aims to identify HNF4A-regulated genes in the intestine and
thus to elucidate the role of this nuclear receptor in intestinal differentiation. As a
model system the authors selected the human cell line Caco-2, one of the best
established models of intestinal differentiation. The authors showed that upon
differentiation HNF4A is up-regulated and thus compared the gene expression
profiles of undifferentiated pre-confluent Caco-2 cells with that of differentiated
post-confluent cells. By combining these gene expression array data with
HNF4A-and histone H3 acetylation-ChIP on chip data of differentiated cells,
actively transcribed genes which are regulated by HNF4A were identified. The
report is in agreement with a previous study performed in hepatocytes and
pancreatic beta-cells, in that a very large number of HNF4A regulated genes
were identified. Three genes, whose regulation by HNF4A was newly identified,
were further investigated: CDX2, trehalase and cingulin. By quantitative PCR of
HNF4A-immunoprecipitated chromatin, gel shift analysis of the putative HNF4A
binding site and promoter reporter studies, the authors showed that these genes
were in fact regulated by HNF4A. Furthermore, the authors confirmed by
quantitative PCR of HNF4A-immunoprecipitated chromatin that the promoters of
CDX2 and trehalase were also bound by HNF4A in the mouse small intestine in
vivo. This was not the case with cingulin.
Altogether, this study is highly interesting, as it fills a gap in the investigation of
the role of the key transcription factor HNF4A: It is the first systematic approach
to identify HNF4A target genes in the intestine. However, several issues require
revision.
Major compulsory revisions
1. It is essential that the authors control for specificity of their ChIP experiments.
In a similar study in hepatocytes, Odom et al., Science 2004 demonstrated by
several lines of evidence the specificity of their results (two different HNF4A
antibodies, preimmune sera etc.). The fact that in 2/3 of potential
HNF4A-regulated promoters, a HNF4A binding site could not be identified by in
silico methods, could thus also be a problem of specificity. How did the authors
control for that problem? The use of pre-immune sera and parallel experiments in
pre-confluent Caco-2 cells is highly recommended.
2. The data showing the results of transfection experiments with
promoter-reporter constructs of CDX2, trehalase and cingulin are all missing.
These are the parts E of figures 2-4! Please give!
Minor essential revisions
1. Page 3, line 3: should read “The cells finally differentiate into the mature cell
types of the intestine.”
2. Page3, last sentence/ first line page 4: Please give a reference for that
statement.
3. Methods: Caco-2 cells are a well established model of intestinal differentiation.
However, it is of paramount interest to give the details of the differentiation
procedure. E.g. were the cells grown on coated or normal dishes, or on filter
insets? Only in the latter case, they build up a tight polarized monolayer and can
be regarded as a model of differentiated human enterocytes. Thus, please
provide a more detailed description of Caco-2 differentiation.
4. Methods, Western Blot, page 6: Please provide the details of nuclear extract
preparation. There are many HNF4A antibodies, which one was used in Western
blotting? GAPDH is not a good control for a constitutive nuclear protein. A better
choice would be e.g. laminin.
5. Methods, ChIP: What was the reason of not choosing an IgG antibody control
for ChIP and instead using an IgG intron amplicon as the negative control in the
quantitative PCR of ChIP? IgG antibody control is the typical ChIP control.
6. Authors’ contributions, page 18: Who has carried out transfection analyses?
7. Legends of Fig. 2-4: Please give the fold molar excess of unlabeled
competitors in gel shift analyses. The legends of parts E is also missing.
Discretionary revisions
1. CDX2 is essential for intestinal differentiation. The authors have shown that it
is regulated by HNF4A. Obviously, this regulation cannot be the cause of
intestinal specific expression of CDX2. The authors may discuss.
2. A comparison of HNF4A-ChIP on chip data between undifferentiated and
differentiated Caco-2 would also allow to more precisely define the role of
HNF4A in differentiation.
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:27 March 2009
Reviewer: Oliver Burk
Reviewer's report:
This manuscripts aims to identify HNF4A-regulated genes in the intestine and
thus to elucidate the role of this nuclear receptor in intestinal differentiation. As a
model system the authors selected the human cell line Caco-2, one of the best
established models of intestinal differentiation. The authors showed that upon
differentiation HNF4A is up-regulated and thus compared the gene expression
profiles of undifferentiated pre-confluent Caco-2 cells with that of differentiated
post-confluent cells. By combining these gene expression array data with
HNF4A-and histone H3 acetylation-ChIP on chip data of differentiated cells,
actively transcribed genes which are regulated by HNF4A were identified. The
report is in agreement with a previous study performed in hepatocytes and
pancreatic beta-cells, in that a very large number of HNF4A regulated genes
were identified. Three genes, whose regulation by HNF4A was newly identified,
were further investigated: CDX2, trehalase and cingulin. By quantitative PCR of
HNF4A-immunoprecipitated chromatin, gel shift analysis of the putative HNF4A
binding site and promoter reporter studies, the authors showed that these genes
were in fact regulated by HNF4A. Furthermore, the authors confirmed by
quantitative PCR of HNF4A-immunoprecipitated chromatin that the promoters of
CDX2 and trehalase were also bound by HNF4A in the mouse small intestine in
vivo. This was not the case with cingulin.
Altogether, this study is highly interesting, as it fills a gap in the investigation of
the role of the key transcription factor HNF4A: It is the first systematic approach
to identify HNF4A target genes in the intestine. However, several issues require
revision.
Major compulsory revisions
1. It is essential that the authors control for specificity of their ChIP experiments.
In a similar study in hepatocytes, Odom et al., Science 2004 demonstrated by
several lines of evidence the specificity of their results (two different HNF4A
antibodies, preimmune sera etc.). The fact that in 2/3 of potential
HNF4A-regulated promoters, a HNF4A binding site could not be identified by in
silico methods, could thus also be a problem of specificity. How did the authors
control for that problem? The use of pre-immune sera and parallel experiments in
pre-confluent Caco-2 cells is highly recommended.
2. The data showing the results of transfection experiments with
promoter-reporter constructs of CDX2, trehalase and cingulin are all missing.
These are the parts E of figures 2-4! Please give!
Minor essential revisions
1. Page 3, line 3: should read “The cells finally differentiate into the mature cell
types of the intestine.”
2. Page3, last sentence/ first line page 4: Please give a reference for that
statement.
3. Methods: Caco-2 cells are a well established model of intestinal differentiation.
However, it is of paramount interest to give the details of the differentiation
procedure. E.g. were the cells grown on coated or normal dishes, or on filter
insets? Only in the latter case, they build up a tight polarized monolayer and can
be regarded as a model of differentiated human enterocytes. Thus, please
provide a more detailed description of Caco-2 differentiation.
4. Methods, Western Blot, page 6: Please provide the details of nuclear extract
preparation. There are many HNF4A antibodies, which one was used in Western
blotting? GAPDH is not a good control for a constitutive nuclear protein. A better
choice would be e.g. laminin.
5. Methods, ChIP: What was the reason of not choosing an IgG antibody control
for ChIP and instead using an IgG intron amplicon as the negative control in the
quantitative PCR of ChIP? IgG antibody control is the typical ChIP control.
6. Authors’ contributions, page 18: Who has carried out transfection analyses?
7. Legends of Fig. 2-4: Please give the fold molar excess of unlabeled
competitors in gel shift analyses. The legends of parts E is also missing.
Discretionary revisions
1. CDX2 is essential for intestinal differentiation. The authors have shown that it
is regulated by HNF4A. Obviously, this regulation cannot be the cause of
intestinal specific expression of CDX2. The authors may discuss.
2. A comparison of HNF4A-ChIP on chip data between undifferentiated and
differentiated Caco-2 would also allow to more precisely define the role of
HNF4A in differentiation.
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:27 March 2009
Reviewer: Oliver Burk
Reviewer's report:
This manuscripts aims to identify HNF4A-regulated genes in the intestine and
thus to elucidate the role of this nuclear receptor in intestinal differentiation. As a
model system the authors selected the human cell line Caco-2, one of the best
established models of intestinal differentiation. The authors showed that upon
differentiation HNF4A is up-regulated and thus compared the gene expression
profiles of undifferentiated pre-confluent Caco-2 cells with that of differentiated
post-confluent cells. By combining these gene expression array data with
HNF4A-and histone H3 acetylation-ChIP on chip data of differentiated cells,
actively transcribed genes which are regulated by HNF4A were identified. The
report is in agreement with a previous study performed in hepatocytes and
pancreatic beta-cells, in that a very large number of HNF4A regulated genes
were identified. Three genes, whose regulation by HNF4A was newly identified,
were further investigated: CDX2, trehalase and cingulin. By quantitative PCR of
HNF4A-immunoprecipitated chromatin, gel shift analysis of the putative HNF4A
binding site and promoter reporter studies, the authors showed that these genes
were in fact regulated by HNF4A. Furthermore, the authors confirmed by
quantitative PCR of HNF4A-immunoprecipitated chromatin that the promoters of
CDX2 and trehalase were also bound by HNF4A in the mouse small intestine in
vivo. This was not the case with cingulin.
Altogether, this study is highly interesting, as it fills a gap in the investigation of
the role of the key transcription factor HNF4A: It is the first systematic approach
to identify HNF4A target genes in the intestine. However, several issues require
revision.
Major compulsory revisions
1. It is essential that the authors control for specificity of their ChIP experiments.
In a similar study in hepatocytes, Odom et al., Science 2004 demonstrated by
several lines of evidence the specificity of their results (two different HNF4A
antibodies, preimmune sera etc.). The fact that in 2/3 of potential
HNF4A-regulated promoters, a HNF4A binding site could not be identified by in
silico methods, could thus also be a problem of specificity. How did the authors
control for that problem? The use of pre-immune sera and parallel experiments in
pre-confluent Caco-2 cells is highly recommended.
2. The data showing the results of transfection experiments with
promoter-reporter constructs of CDX2, trehalase and cingulin are all missing.
These are the parts E of figures 2-4! Please give!
Minor essential revisions
1. Page 3, line 3: should read “The cells finally differentiate into the mature cell
types of the intestine.”
2. Page3, last sentence/ first line page 4: Please give a reference for that
statement.
3. Methods: Caco-2 cells are a well established model of intestinal differentiation.
However, it is of paramount interest to give the details of the differentiation
procedure. E.g. were the cells grown on coated or normal dishes, or on filter
insets? Only in the latter case, they build up a tight polarized monolayer and can
be regarded as a model of differentiated human enterocytes. Thus, please
provide a more detailed description of Caco-2 differentiation.
4. Methods, Western Blot, page 6: Please provide the details of nuclear extract
preparation. There are many HNF4A antibodies, which one was used in Western
blotting? GAPDH is not a good control for a constitutive nuclear protein. A better
choice would be e.g. laminin.
5. Methods, ChIP: What was the reason of not choosing an IgG antibody control
for ChIP and instead using an IgG intron amplicon as the negative control in the
quantitative PCR of ChIP? IgG antibody control is the typical ChIP control.
6. Authors’ contributions, page 18: Who has carried out transfection analyses?
7. Legends of Fig. 2-4: Please give the fold molar excess of unlabeled
competitors in gel shift analyses. The legends of parts E is also missing.
Discretionary revisions
1. CDX2 is essential for intestinal differentiation. The authors have shown that it
is regulated by HNF4A. Obviously, this regulation cannot be the cause of
intestinal specific expression of CDX2. The authors may discuss.
2. A comparison of HNF4A-ChIP on chip data between undifferentiated and
differentiated Caco-2 would also allow to more precisely define the role of
HNF4A in differentiation.
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