|
Graphical Abstract
|
|
Figure 1. Gata4 conditional knock-in mice express GATA4 in the ileum. (A) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT-Gata4. The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence (loxP-PGK-Neo-3×SV40pA-loxP, LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT-Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA-Gata4. Homologous recombination between pROSA26PA-Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sortm1(Gata4)Bat, designated ROSA26lnlG4. After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI (B) and EcoRV (E) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers (Table 1, primers). (B) Southern blot analysis confirmed germline transmission of the ROSA26lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26+/+) or a mouse heterozygous for the modified ROSA26 allele (ROSA26lnlG4/+). We observed the expected fragments representing the wild-type and modified alleles (EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). (C) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26lnlG4/+Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice (ROSA26lnlG4/+). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: *P ≤ .05. (D) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars: 100 μm. (E) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: *P ≤ .05, **P ≤ .001.
|
|
Figure 2. Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. (A) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars: 100 μm. (B) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26lnlG4/+Villin-Cre (designated Gata4 cKI) mice compared with control mice (ROSA26lnlG4/+) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. (C) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.
|
|
Figure 3. Identification of high-confidence GATA4 targets that define jejunal vs ileal enterocyte identity. (A) We used Affymetrix oligonucleotide array analysis with RNA from wild-type (WT) CD-1 duodenal, jejunal, and ileal epithelial cells (n = 3) to identify sets of regionally enriched transcripts and with RNA from ileal epithelial cells of control (Rosa26lnlG4/+) and Gata4 cKI (Rosa26lnlG4/+Villin-Cre) mice (n = 3 per genotype) to identify transcripts altered in GATA4-expressing (GATA+) ileum compared with control ileum. A threshold of at least a 2-fold change and an unadjusted P value ≤ .05 was used for comparisons. We determined overlap between transcripts differentially expressed in GATA4+ ileum (136 transcripts) and those identified as either duodenal-enriched, jejunal/duodenal-enriched, jejunal-enriched, or ileal-enriched (bolded text). We found that 47% of transcripts with altered expression in GATA4+ ileum overlapped with proximal-enriched (duodenal, jejunal/duodenal, and jejunal sets, indicated by bracket) or ileal-enriched transcript sets (64 of 136, 32 proximal-enriched, 32 ileal-enriched). (B) Of the 32 proximal-enriched transcripts identified, the expression of 21 was increased in GATA4+ ileum. Of the 32 ileal-enriched transcripts identified, the expression of 29 was decreased in GATA4+ ileum. (C) Focusing on the jejunal-enriched set (17 transcripts, including 15 jejunal-enriched and 12 jejunal/duodenal-enriched) and the ileal-enriched set (29 transcripts), we reasoned that high-confidence GATA4 direct targets would show converse expression changes in the presence or absence of GATA. Therefore, to identify transcripts differentially expressed in GATA4-deficient jejunum, we used Affymetrix oligonucleotide array analysis with RNA from jejunal epithelial cells of Gata4 cKO (Gata4loxP/loxPVillin-Cre) mice (n = 3) and WT CD-1 mice (n = 3, same arrays as in panel A). A threshold of at least a 2-fold change and an unadjusted P value ≤ .05 was used. We determined overlap between the 46 jejunal- or ileal-enriched transcripts and those transcripts differentially expressed in GATA4-deficient jejunum (466 transcripts). We found that 15 of 17 jejunal-enriched transcripts with increased expression in GATA4+ ileum and 18 of 29 ileal-enriched transcripts with decreased expression in GATA4+ ileum overlapped with those altered in GATA4-deficient jejunum and that all 33 overlapping transcripts showed a converse expression pattern in GATA4-deficient jejunum compared with GATA4+ ileum (ie, expression of all 15 jejunal-enriched transcripts was decreased in GATA4-deficient jejunum and expression of all 18 ileal-enriched transcripts was increased in GATA4-deficient jejunum). Specific jejunal- and ileal-enriched transcripts are listed in the box. Those bolded are important in the enterohepatic circulation pathway.
|
|
Figure 4. GATA4-expressing ileum loses ileal identity. Transcripts identified as having enriched expression in the epithelium of duodenum, jejunum/duodenum, jejunum, or ileum of wild-type animals (Figure 3A and Supplementary Table 1) were used as reference gene sets in gene set enrichment analysis. CEL files for microarrays performed with RNA isolated from ileal epithelial cells of control (Rosa26lnlG4/+) and Gata4 cKI (Rosa26lnlG4/+Villin-Cre) mice (n = 3 per genotype) were tested for enrichment of gene sets by gene set enrichment analysis. The top panels and bottom left panel show gene set enrichment analysis using transcripts enriched in duodenal (top left), jejunal/duodenal (top right), or jejunal (bottom left) epithelium as the reference gene set; the bottom left panel shows gene set enrichment analysis using transcripts enriched in ileal epithelium as the reference gene set. Proximal transcripts were highly enriched (normalized enrichment score [NES] = +2.27, +2.84, and +1.92) in GATA4-expressing (GATA4+) ileum, whereas ileal transcripts were enriched in control ileum (NES = -3.63). All were statistically significant by P value and false-discovery rate (FDR).
|
|
Figure 5. Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI (ROSA26lnlG4/+Villin-Cre) and control (ROSA26lnlG4/+) mice and in jejunal epithelial cells from Gata4 cKO (Gata4loxP/loxPVillin-Cre) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold. Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. *P ≤ .05. **P ≤ .01.
|
|
Figure 6. GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum (top panel) and within genes repressed in jejunum (middle panel). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites (Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice (Cdk4) or absent in ileum of control and Gata4 cKI mice (Alb) (bottom panel). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7. Enrichment per sample was normalized to input (n = 6 Gata4flbio/flbio::ROSA26BirA/BirA mice, designated GATA4-FlagBio/BirA, and 6 ROSA26BirA/BirA mice, designated GATA4-WT/BirA). Error bars show SEM. P values were determined by 2-sample Student t test: *P ≤ .05, **P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene (Slc10a2_1 and Slc10a2_2) was analyzed previously by qPCR.38
|
|
Figure 7. Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls (Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.
|
|
Figure 8. GATA4 binds to GATA consensus binding sites in jejunal-enriched genes and in ileal-enriched genes regulated by GATA4 expression in the intestine. (A) Table shows double-stranded oligonucleotide EMSA probes (5’ to 3’). Underlined sequences identify the location of GATA4 consensus binding sites within probes. Nucleotides mutated to eliminate GATA4 consensus binding sites in mutant probes are indicated. Two distinct probes were designed for the Fgf15 gene because there were 2 consensus GATA4 binding sites within approximately 100 bp of each other within the peak of interest. (B) Immunoblot analysis of nuclear extracts from 293T cells transfected with either control empty plasmid or GATA4 containing plasmid (pcDNA-GATA4) shows overexpression of GATA4 in pcDNA-GATA4–transfected cells. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. Molecular weight marker locations are indicated. (C) Representative EMSA competition assay is shown. Nuclear extract from pcDNA-GATA4–transfected 293T cells was incubated with radiolabeled xiFABP probe in the absence (lane 1) or presence (lanes 2–11) of 200-fold molar excess of unlabeled competitors. A bracket indicates the location of GATA4-bound xiFABP probe. Data are representative of 3 independent assays.
|
|
Figure 9. Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. (A) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR (Figure 7) in jejunal epithelial cells from control and Gata4 cKI mice. (B) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. (A and B) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.
|
|
Figure 10. Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. (A) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel, and 7 of 14 low to no SLC10A2 staining, lower panel). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. (B) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice (ROSA26lnlG4/+Villin-Cre) was 9% of the level observed in control ileum (ROSA26lnlG4/+; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: *P ≤ .05. (C) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice (ROSA26lnlG4/+Villin-Cre) was 26% and 19% of the level observed in control ileum (ROSA26lnlG4/+), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: *P ≤ .05. (D) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice (ROSA26lnlG4/+Villin-Cre) compared with control mice (ROSA26lnlG4/+; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. *P ≤ .05.
|
|
Figure 11. Lactase protein is expressed in Gata4 cKI ileum. (A) Immunofluorescent staining showed Lactase protein (LCT, green) at the brush border in ileal epithelium of Gata4 cKI mice, whereas LCT protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. E-cadherin (ECAD, red) was used to stain epithelial cell membranes. 4′,6-diamidino-2-phenylindole (DAPI) (blue) stained nuclei. Scale bars: 100 μm. (B) Immunoblot analysis of protein extracts from ileal epithelial cells of control and Gata4 cKI mice was used to quantify LCT protein (n = 3). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. Arrowhead indicates the LCT band measured. LCT protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: *P ≤ .05.
|
|
|
