BACKGROUND
the mature mammalian small intestine is lined by a highly specialized epithelium that regenerates itself in a tightly controlled manner resulting in a lineage distribution and gene expression patterning that is perfectly suited for the absorption of nutrients.
the epithelium is organized into crypt-villus structures in which the crypts of lieberkühn contain stem cells that produce proliferating, transit-amplifying  cells that differentiate into five principal post-mitotic cell types comprised of one type of absorptive cell  and four types of secretory cells .
absorptive enterocytes express digestive enzymes and transporters necessary for the terminal digestion and absorption of nutrients.
mucus-secreting goblet cells and defensin-secreting paneth cells maintain a dynamic mucosal defensive barrier.
enteroendocrine cells secrete hormones that regulate gastrointestinal processes.
tuft cells, recently shown to be an independent secretory lineage  <cit> , secrete opioids and produce enzymes that synthesize prostaglandins, suggesting a role in inflammation.
absorptive enterocytes, goblet cells, enteroendocrine cells, and tuft cells migrate up the crypt to populate the villi, whereas paneth cells migrate to the base of crypts.
the differentiated cells eventually undergo apoptosis and are shed into the lumen.
cells of the villus epithelium turn over in 3–4 days, whereas paneth cells at the base of crypts turn over at a slower rate of 3–6 weeks.
current models of intestinal epithelial renewal suggest that long-lived, multipotent stem cells produce progenitors that undergo a series of transitions that ultimately give rise to the individual cell lineages  <cit> .
the wnt, hedgehog, and bone morphogenetic protein signaling pathways regulate intestinal proliferation and differentiation, while the notch signaling pathway plays a central role in determining epithelial cell fate.
the first decision selects absorptive vs. secretory progenitors.
activated notch signaling results in the transcriptional activation of its principal intestinal target, hairy and enhancer of split  <dig> , which encodes a transcription factor that selects the absorptive enterocyte lineage.
progenitor cells that escape notch signaling and activation of hes <dig> gene transcription express atonal homolog  <dig> , which encodes a transcription factor that selects the secretory cells.
additional regulators that function in secretory cell differentiation include: growth factor independent  <dig>  that distinguishes enteroendocrine from goblet/paneth progenitors; neurogenin  <dig>  that specifies the enteroendocrine lineage; sam pointed domain-containing ets transcription factor , a gfi <dig> target, that promotes goblet differentiation; and sry-box containing gene  <dig>  and ephrin type b receptor  <dig> , wnt targets that are necessary for the differentiation of paneth cells and their localization to the crypt base, respectively.
recently, we showed that members of the gata family, an ancient family of transcription factors that bind wgatar motifs in dna, play essential roles in crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression.
gata <dig> and gata <dig> are expressed in the intestinal epithelium, but whereas gata <dig> is expressed throughout the length of the small intestine, gata <dig> is expressed in the proximal 85% of small intestine and is sharply down-regulated in the distal ileum  <cit> .
using conditional knockout technology, we  <cit>  and others  <cit>  have shown that gata <dig> functions to promote a 'jejunal’ pattern of absorptive enterocyte gene expression and function while repressing an 'ileal’ pattern.
using single and double conditional knockout approaches for gata <dig> and gata <dig>  we found that in the ileum of single gata <dig> conditional knockout mice, where gata <dig> is not normally expressed, or throughout the small intestine of double gata4/gata <dig> conditional knockout mice, crypt cell proliferation and enteroendocrine cell specification are decreased, paneth cells are replaced by a goblet-like cell type, and the expression of specific absorptive enterocyte genes is altered  <cit> .
we also noted that spdef, a transcription factor expressed in secretory progenitors, goblet cells and paneth cells that functions in goblet and paneth cell differentiation  <cit> , was up-regulated  <cit> .
using a conditional spdef over-expression model, noah et al.
<cit>  described an intestinal phenotype that, with the exception of the changes in absorptive enterocyte gene expression, essentially phenocopies that of our gata4/gata <dig> conditional knockout mice: crypt cell proliferation is decreased, enteroendocrine and paneth cells are decreased, and goblet-like cells are increased.
gata <dig> is co-expressed with spdef in the same lineages in the small intestine  <cit> .
based on these findings  <cit> , we hypothesized that gata4/gata <dig> regulates crypt cell proliferation and secretory cell differentiation in the small intestine by repressing spdef gene expression.
to test this hypothesis, we defined the functions most likely regulated by the overlapping gata6/spdef target gene set in mouse intestine, delineated the relationship between gata <dig> chromatin occupancy and spdef gene regulation in caco- <dig> cells, and determined the extent to which prevention of spdef up-regulation by spdef knockout rescues the gata <dig> phenotype in conditional gata <dig> knockout mouse ileum.
RESULTS
gata <dig> and spdef regulate similar subsets of genes
to gain insight on the relationship between gata <dig> and spdef in the small intestine, we scanned the overlap of gene targets using publicly available gene profiling data from conditional gata <dig> and spdef knockout mouse intestine  <cit> .
previously, we identified  <dig> genes whose expression is altered in ileum by conditional gata <dig> deletion  <cit> .
network analysis of this gene set indicated an up-regulation of p <dig> targets and a down-regulation of c-myc targets , consistent with a decrease in cellular proliferation.
of the  <dig> genes altered by conditional gata <dig> knockout, 83%  were also altered by spdef knockout , a far greater overlap than would be expected from a similar-sized, randomized allocation of genes .
the changes in expression of this subset when gata <dig> is deleted were analyzed by gene set array analysis.
using database for annotation, visualization and integrated discovery , we conducted functional annotation clustering of all the major pathways .
we found that wnt signaling was the function most likely to be effected , consistent with regulation of crypt cell proliferation.
using gene set enrichment analysis , a publically available bioinformatics tool that delineates gene expression data for enrichment of pre-defined gene-sets, apc target network was one of the top three networks affected by gata <dig> deletion with a normalized enrichment score  of - <dig>  .
this analysis reveals a very strong overlap in gene targets between gata <dig> and spdef, and suggests that the principal function of this overlap involves cellular proliferation.
gata <dig> regulates spdef gene expression and occupies a locus in the spdef 5′-flanking region in human caco- <dig> cells
conditional gata <dig> deletion in mice produces an up-regulation of spdef gene expression in ileum while knockout or over-expression of spdef has no effect on gata <dig> expression  <cit> , suggesting that spdef is regulated downstream by gata <dig>  to determine the extent to which this process is conserved, and to further explore the role of gata <dig> in regulating spdef gene expression, we characterized the effect of gata <dig> knock-down on spdef gene expression in caco- <dig> cells.
the caco- <dig> cell line is a human colorectal adenocarcinoma-derived cell line that expresses gata <dig>  but very little gata <dig>  <cit> , similar to the ileum.
we screened five gata <dig> short-hairpin rna  knockdown lentiviral constructs, and found that only one resulted in a statistically significant knockdown of gata <dig> mrna  and a concomitant decrease in gata <dig> protein .
spdef mrna was up-regulated 2-fold in gata <dig> knock-down  cells , demonstrating that the caco- <dig> cells model the up-regulation of spdef gene expression observed in conditional gata <dig> knockout mouse ileum  <cit> .
to gain insight on the relationship between gata <dig> and spdef gene expression, we examined gata <dig> chromatin occupancy at the spdef gene locus.
because we were unable to immunoprecipitate bound chromatin from crosslinked mouse intestinal epithelial cells using existing antisera, we utilized the human caco- <dig> intestinal cell culture model in which gata <dig> chip assays have been performed previously  <cit> .
using a publicly available gata <dig> chromatin immunoprecipitation-high throughput sequencing  database in caco- <dig> cells  <cit> , we identified a gata6-occupied cis-regulatory region approximately 40 kb upstream of the spdef transcription start site  that mapped to the spdef gene based on closest distance to any tss .
to confirm and localize gata <dig> occupancy at this locus, we performed gata <dig> chip assays using a multiple, overlapping primer strategy with amplicons of ~200 bp per primer pair  centered at the only gata motif within this region.
we found increases of all three amplicons with amplicon from primer set  <dig> being significantly greater  than the amy <dig> tss negative control .
although our chip data reveal a modest increase in enrichment, it is nonetheless statistically significant and thus confirmatory of chip-seq data conducted by others  <cit>  .
we also conducted an igg control chip assay to show that enrichment with primer set  <dig> was not due to differences in primer efficiency from that of the amy <dig> tss, or to non-specific antibody binding .
these data confirm previous chip-seq analysis that gata <dig> occupies a cis-regulatory region that maps to the spdef gene  <cit> , and, together with spdef up-regulation in g6kd cells, is consistent with the notion that gata <dig> directly represses spdef gene transcription.
while mapping of occupancy sites to the nearest tss is a well recognized method for defining transcription factor targets on a global basis  <cit> , direct regulation of spdef gene expression by gata <dig> at this site will need to be confirmed using chromosome conformation capture or other techniques  <cit> .
transcriptional repression is highly complex, especially in eukaryotes, generally involving the recruitment of specific co-repressors and the local modification of histone tails and chromatin structure  <cit> .
gata factors are well known to mediate gene repression, and the mechanisms are beginning to be understood.
gata <dig> has been shown to directly repress cardiac genes  <cit>  while gata <dig> has been shown to directly repress hematopoietic genes  <cit> .
both gata <dig> and gata <dig> interact directly with corepressor complexes including the nucleosome remodeling and histone deacetylase  complex and the polycomb repressive complex  <dig>   <cit> , and are necessary for the multiple modifications of histone tails, but the extent to which gata factors recruit co-repressors and/or modulate histone tails in the small intestine remains to be determined.
spdef knockout rescues the proliferation defect in conditional gata <dig> knockout mice
we next asked to what extent spdef deletion rescues the conditional gata <dig> knockout phenotype by analyzing single and double gata6/spdef knockout mice.
in previous studies, conditional deletion of gata <dig> <cit>  or deletion of spdef <cit>  resulted in greatly diminished levels of gata <dig> or spdef mrna, respectively, in the intestine that correlated with reduced protein levels and altered intestinal phenotypes.
analysis of mrna in the present study showed that gata <dig> and spdef mrnas were both expressed in ctl mice, gata <dig> mrna was nearly undetectable while spdef mrna was expressed in gata6Δie mice, gata <dig> mrna was expressed while spdef mrna was nearly undetectable in spdefko mice, and both gata <dig> and spdef mrnas were nearly undetectable in the double knockout  mice , verifying our models.
crypt cell proliferation is essential for the continuation of intestinal epithelial renewal.
one of the principal phenotypic outcomes of conditional gata4/gata <dig> deletion is a ~30% reduction in the number of ki67- and brdu-positive cells, and a concomitant decrease in villus height and villus epithelial cell number  <cit>  resulting in a reduction in absorptive surface area.
to define the role of spdef in mediating the decrease in crypt cell proliferation when gata <dig> is conditionally deleted, we stained ileal segments for ki <dig> and brdu , markers of the non-g <dig> and s-phases of the cell cycle, respectively.
the number of ki67-positive and brdu-positive crypt cells was significantly reduced ~40% in gata6Δie mice , as previously reported  <cit> , but unchanged in spdefko mice, as compared to controls.
the number of ki67- and brdu-positive crypt cells in dko mice was also similar to controls, indicating that the additional deletion of spdef rescues the decrease in crypt cell proliferation observed in gata6Δie mice.
these data support the notion that gata <dig> maintains crypt cell proliferation by down-regulating spdef gene expression.
spdef does not regulate the secretory cell differentiation function of gata6
previously, conditional deletion of gata <dig> in the ileum resulted in a 25-40% reduction in the number of chromogranin a -positive cells, and in the mrna abundances of chga and neurog <dig> <cit> , consistent with a decrease in enteroendocrine lineage commitment.
though not statistically significant, we found a 15-50% reduction in the number of chga-positive cells , and in the mrna abundances of chga and neurog <dig>  in the gata6Δie mice, in general agreement with our previous study  <cit> .
we further found that although the pattern for these three measurements in the spdefko mice was similar to that in ctl mice, consistent with previous data for distal intestine  <cit> , the pattern in the dko mice was similar to that in the gata6Δie mice .
together, these data show that enteroendocrine cell commitment is reduced in both gata6Δie and dko mice, indicating that gata <dig> promotes enteroendocrine cell commitment independently of spdef.
previously, we also showed that conditional gata <dig> deletion resulted in a transformation of paneth cells into mucin- <dig> -enriched goblet-like cells at the base of crypts  <cit> .
in the present study, immunostaining for crs4c, a paneth-specific marker, was slightly reduced in spdefko mice as compared to controls, consistent with previous data  <cit> , but was greatly reduced in both gata6Δie and dko mice as compared to controls .
the number of crypt cross-sections with at least one crs4c-positive cell was nearly 100% in ctl and spdefko mice, but was less than 30% in both gata6Δie and dko mice .
the mrna abundance for lysozyme , another paneth marker, was not significantly different between ctl and spdefko mice, but was significantly reduced in gata6Δie and dko mice as compared to controls .
pas staining was greatly increased in crypts of gata6Δie mice, as previously shown  <cit> , noticeably reduced in spdefko mice, especially in villus goblet cells, as previously shown  <cit> , and similar to controls in dko mice .
muc <dig> immunostaining was greatly enriched in crypts of gata6Δie mice, consistent with our previous report  <cit> , but similar to controls in spdefko and dko mice .
generally consistent with this observation, muc <dig> mrna abundance in gata6Δie mice was  <dig> -fold higher as compared to controls, though not statistically significant, whereas muc <dig> mrna abundances in spdefko and dko mice were similar to controls .
these data indicate that the additional deletion of spdef did not rescue the decrease in the terminal differentiation of paneth cells observed in gata6Δie mice, but did at least partially prevent their conversion to muc2-enriched goblet-like cells.
thus, spdef is not necessary for the gata-mediated differentiation of paneth cells, but does function in their default differentiation into goblet-like cells in the absence of gata <dig>  this is consistent with the general function of spdef to promote the differentiation of goblet cells  <cit> .
in spite of the common secretory cell phenotypes in gata <dig> deletion and spdef over-expression models  <cit> , our data show that spdef deletion did not rescue the gata <dig> knockout defects in enteroendocrine lineage commitment or in paneth cell differentiation.
closer scrutiny suggests that the secretory cell phenotypes in the gata <dig> deletion and spdef over-expression models are not identical.
while the decline in enteroendocrine and paneth cells, and accumulation of goblet cells, appear similar, the underlying alteration in paneth and goblet cell differentiation is different.
conditional gata <dig> deletion has no effect on the normal commitment and differentiation of goblet cells on villi but reveals a conversion of paneth cells at the base of crypts into a goblet-like cell type.
these cells do not express defensins, but express abundant muc2; they also express abundant sox <dig> and ephb <dig> <cit> , indicating that they are committed and targeted paneth cells.
on the other hand, conditional spdef over-expression results in a generalized increase in goblet cells at the expense of all other epithelial cell types  <cit> .
hence, these mice show a decline in paneth cell specification rather than a defect in the terminal differentiation of committed paneth cells, as observed in our conditional gata <dig> knockout model.
spdef does not regulate the absorptive enterocyte gene expression function of gata6
we next examined whether spdef loss affected gata6-dependent absorptive enterocyte gene expression.
conditional gata <dig> deletion resulted in a down-regulation of specific absorptive enterocyte genes in ileum that include lipid transporters and apolipoproteins, and an up-regulation of genes in absorptive enterocytes normally not expressed or expressed at low levels in small intestine, but expressed at high levels in colon  <cit> .
using marker genes for these two patterns, apolipoprotein a <dig>  and carbonic anyhdrase  <dig> , respectively, we found that their mrna abundances were down-regulated and up-regulated, respectively, in gata6Δie mice , consistent with our previous findings  <cit> .
apoa <dig> and car <dig> mrna abundance in spdefko mice was similar to controls, whereas that in dko mice was similar to gata6Δie, indicating that spdef does not play a role in mediating the conditional gata <dig> knockout alteration in absorptive enterocyte gene expression.
gata <dig> is expressed in crypts and in mature absorptive enterocytes  <cit> , whereas spdef is expressed in secretory progenitors, and in goblet and paneth cells, but is not expressed in mature absorptive enterocytes  <cit> .
while it is possible that spdef could instruct gene expression in absorptive enterocytes through a process that originates in progenitors early in the differentiation process, we found that spdef did not regulate the gata <dig> targets studied here.
as in the gata4-specific pathway, in which gata <dig> regulates its targets in mature absorptive enterocytes on villi rather than in crypt progenitors  <cit> , we believe that gata <dig> also regulates absorptive enterocyte gene expression within mature absorptive enterocytes on villi.
CONCLUSIONS
previously, we defined two fundamental pathways of gata regulation in the small intestine, one mediated exclusively by gata <dig> , and one regulated by gata <dig> or gata <dig>  .
in the gata4-specific pathway, gata <dig>  but not gata <dig>  activates and represses a subset of absorptive enterocyte genes, and by virtue of its expression in the proximal 85% of small intestine and lack of expression in distal ileum  <cit> , distinguishes proximal intestinal from distal ileal gene expression and function  <cit> .
for the gata4/gata6-redundant pathway, either gata <dig> or gata <dig>  promote intestinal epithelial renewal by supporting crypt cell proliferation, enteroendocrine lineage commitment, paneth cell differentiation, and absorptive enterocyte gene expression  <cit> .
here, we show that the maintenance of crypt cell proliferation function of the intestinal gata4/gata6-redundant pathway is dependent on spdef .
specifically, our data support the notion that gata4/gata <dig> promotes crypt cell proliferation by directly repressing spdef gene expression.
gata <dig> has been suggested to be oncogenic in multiple cancers and pre-cancerous lesions, including barrett’s esophagus, gastric cancer, pancreatic cancer, and colon cancer  <cit> .
gata <dig> expression is up-regulated in colon cancer epithelial cells  <cit> , as well as in non-malignant cells along the stromal margins in human colorectal cancer  <cit> , but it should be noted that it has not been determined whether this is a correlative, causative or protective event.
recently, noah et al.
<cit>  showed that spdef functions as a colorectal tumor suppressor.
in colorectal tumors from patients, loss of spdef was observed in approximately 85% of tumors and correlated with progression from normal tissue, to adenoma, to adenocarcinoma.
further, spdef inhibited the expression of β-catenin-target genes in mouse colon tumors, and interacted with β-catenin to block its transcriptional activity in colorectal cancer cell lines, resulting in lower levels of cyclin d <dig> and c-myc.
pathway analysis showed that gata <dig> deletion also modulated c-myc targets  and that the gata6/spdef overlapping gene set likely functions in wnt signaling .
thus, our data here suggest that the possible tumorigenic effects of gata <dig> could be mediated by its repression of spdef gene expression.

 the mature mammalian small intestine is lined by a highly specialized epithelium that regenerates itself in a tightly controlled manner resulting in a lineage distribution and gene expression patterning that is perfectly suited for the absorption of nutrients. the epithelium is organized into crypt-villus structures in which the crypts of lieberkühn contain stem cells that produce proliferating, transit-amplifying cells that differentiate into five principal post-mitotic cell types comprised of one type of absorptive cell and four types of secretory cells. absorptive enterocytes express digestive enzymes and transporters necessary for the terminal digestion and absorption of nutrients. mucus-secreting goblet cells and defensin-secreting paneth cells maintain a dynamic mucosal defensive barrier. enteroendocrine cells secrete hormones that regulate gastrointestinal processes. tuft cells, recently shown to be an independent secretory lineage, secrete opioids and produce enzymes that synthesize prostaglandins, suggesting a role in inflammation. absorptive enterocytes, goblet cells, enteroendocrine cells, and tuft cells migrate up the crypt to populate the villi, whereas paneth cells migrate to the base of crypts. the differentiated cells eventually undergo apoptosis and are shed into the lumen. cells of the villus epithelium turn over in 3–4 days, whereas paneth cells at the base of crypts turn over at a slower rate of 3–6 weeks. current models of intestinal epithelial renewal suggest that long-lived, multipotent stem cells produce progenitors that undergo a series of transitions that ultimately give rise to the individual cell lineages. the wnt, hedgehog, and bone morphogenetic protein signaling pathways regulate intestinal proliferation and differentiation, while the notch signaling pathway plays a central role in determining epithelial cell fate. the first decision selects absorptive vs. secretory progenitors. activated notch signaling results in the transcriptional activation of its principal intestinal target, hairy and enhancer of split, which encodes a transcription factor that selects the absorptive enterocyte lineage. progenitor cells that escape notch signaling and activation of hes gene transcription express atonal homolog, which encodes a transcription factor that selects the secretory cells. additional regulators that function in secretory cell differentiation include: growth factor independent that distinguishes enteroendocrine from goblet/paneth progenitors; neurogenin that specifies the enteroendocrine lineage; sam pointed domain-containing ets transcription factor, a gfi target, that promotes goblet differentiation; and sry-box containing gene and ephrin type b receptor, wnt targets that are necessary for the differentiation of paneth cells and their localization to the crypt base, respectively. recently, we showed that members of the gata family, an ancient family of transcription factors that bind wgatar motifs in dna, play essential roles in crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression. gata and gata are expressed in the intestinal epithelium, but whereas gata is expressed throughout the length of the small intestine, gata is expressed in the proximal 85% of small intestine and is sharply down-regulated in the distal ileum. using conditional knockout technology, we and others have shown that gata functions to promote a'jejunal’ pattern of absorptive enterocyte gene expression and function while repressing an'ileal’ pattern. using single and double conditional knockout approaches for gata and gata we found that in the ileum of single gata conditional knockout mice, where gata is not normally expressed, or throughout the small intestine of double gata4/gata conditional knockout mice, crypt cell proliferation and enteroendocrine cell specification are decreased, paneth cells are replaced by a goblet-like cell type, and the expression of specific absorptive enterocyte genes is altered. we also noted that spdef, a transcription factor expressed in secretory progenitors, goblet cells and paneth cells that functions in goblet and paneth cell differentiation, was up-regulated. using a conditional spdef over-expression model, noah et al. described an intestinal phenotype that, with the exception of the changes in absorptive enterocyte gene expression, essentially phenocopies that of our gata4/gata conditional knockout mice: crypt cell proliferation is decreased, enteroendocrine and paneth cells are decreased, and goblet-like cells are increased. gata is co-expressed with spdef in the same lineages in the small intestine. based on these findings, we hypothesized that gata4/gata regulates crypt cell proliferation and secretory cell differentiation in the small intestine by repressing spdef gene expression. to test this hypothesis, we defined the functions most likely regulated by the overlapping gata6/spdef target gene set in mouse intestine, delineated the relationship between gata chromatin occupancy and spdef gene regulation in caco- cells, and determined the extent to which prevention of spdef up-regulation by spdef knockout rescues the gata phenotype in conditional gata knockout mouse ileum.  gata and spdef regulate similar subsets of genes to gain insight on the relationship between gata and spdef in the small intestine, we scanned the overlap of gene targets using publicly available gene profiling data from conditional gata and spdef knockout mouse intestine. previously, we identified genes whose expression is altered in ileum by conditional gata deletion. network analysis of this gene set indicated an up-regulation of p targets and a down-regulation of c-myc targets, consistent with a decrease in cellular proliferation. of the genes altered by conditional gata knockout, 83% were also altered by spdef knockout, a far greater overlap than would be expected from a similar-sized, randomized allocation of genes. the changes in expression of this subset when gata is deleted were analyzed by gene set array analysis. using database for annotation, visualization and integrated discovery, we conducted functional annotation clustering of all the major pathways. we found that wnt signaling was the function most likely to be effected, consistent with regulation of crypt cell proliferation. using gene set enrichment analysis, a publically available bioinformatics tool that delineates gene expression data for enrichment of pre-defined gene-sets, apc target network was one of the top three networks affected by gata deletion with a normalized enrichment score of -. this analysis reveals a very strong overlap in gene targets between gata and spdef, and suggests that the principal function of this overlap involves cellular proliferation. gata regulates spdef gene expression and occupies a locus in the spdef 5′-flanking region in human caco- cells conditional gata deletion in mice produces an up-regulation of spdef gene expression in ileum while knockout or over-expression of spdef has no effect on gata expression, suggesting that spdef is regulated downstream by gata to determine the extent to which this process is conserved, and to further explore the role of gata in regulating spdef gene expression, we characterized the effect of gata knock-down on spdef gene expression in caco- cells. the caco- cell line is a human colorectal adenocarcinoma-derived cell line that expresses gata but very little gata, similar to the ileum. we screened five gata short-hairpin rna knockdown lentiviral constructs, and found that only one resulted in a statistically significant knockdown of gata mrna and a concomitant decrease in gata protein. spdef mrna was up-regulated 2-fold in gata knock-down cells, demonstrating that the caco- cells model the up-regulation of spdef gene expression observed in conditional gata knockout mouse ileum. to gain insight on the relationship between gata and spdef gene expression, we examined gata chromatin occupancy at the spdef gene locus. because we were unable to immunoprecipitate bound chromatin from crosslinked mouse intestinal epithelial cells using existing antisera, we utilized the human caco- intestinal cell culture model in which gata chip assays have been performed previously. using a publicly available gata chromatin immunoprecipitation-high throughput sequencing database in caco- cells, we identified a gata6-occupied cis-regulatory region approximately 40 kb upstream of the spdef transcription start site that mapped to the spdef gene based on closest distance to any tss. to confirm and localize gata occupancy at this locus, we performed gata chip assays using a multiple, overlapping primer strategy with amplicons of ~200 bp per primer pair centered at the only gata motif within this region. we found increases of all three amplicons with amplicon from primer set being significantly greater than the amy tss negative control. although our chip data reveal a modest increase in enrichment, it is nonetheless statistically significant and thus confirmatory of chip-seq data conducted by others. we also conducted an igg control chip assay to show that enrichment with primer set was not due to differences in primer efficiency from that of the amy tss, or to non-specific antibody binding. these data confirm previous chip-seq analysis that gata occupies a cis-regulatory region that maps to the spdef gene, and, together with spdef up-regulation in g6kd cells, is consistent with the notion that gata directly represses spdef gene transcription. while mapping of occupancy sites to the nearest tss is a well recognized method for defining transcription factor targets on a global basis, direct regulation of spdef gene expression by gata at this site will need to be confirmed using chromosome conformation capture or other techniques. transcriptional repression is highly complex, especially in eukaryotes, generally involving the recruitment of specific co-repressors and the local modification of histone tails and chromatin structure. gata factors are well known to mediate gene repression, and the mechanisms are beginning to be understood. gata has been shown to directly repress cardiac genes while gata has been shown to directly repress hematopoietic genes. both gata and gata interact directly with corepressor complexes including the nucleosome remodeling and histone deacetylase complex and the polycomb repressive complex, and are necessary for the multiple modifications of histone tails, but the extent to which gata factors recruit co-repressors and/or modulate histone tails in the small intestine remains to be determined. spdef knockout rescues the proliferation defect in conditional gata knockout mice we next asked to what extent spdef deletion rescues the conditional gata knockout phenotype by analyzing single and double gata6/spdef knockout mice. in previous studies, conditional deletion of gata or deletion of spdef resulted in greatly diminished levels of gata or spdef mrna, respectively, in the intestine that correlated with reduced protein levels and altered intestinal phenotypes. analysis of mrna in the present study showed that gata and spdef mrnas were both expressed in ctl mice, gata mrna was nearly undetectable while spdef mrna was expressed in gata6Δie mice, gata mrna was expressed while spdef mrna was nearly undetectable in spdefko mice, and both gata and spdef mrnas were nearly undetectable in the double knockout mice, verifying our models. crypt cell proliferation is essential for the continuation of intestinal epithelial renewal. one of the principal phenotypic outcomes of conditional gata4/gata deletion is a ~30% reduction in the number of ki67- and brdu-positive cells, and a concomitant decrease in villus height and villus epithelial cell number resulting in a reduction in absorptive surface area. to define the role of spdef in mediating the decrease in crypt cell proliferation when gata is conditionally deleted, we stained ileal segments for ki and brdu, markers of the non-g and s-phases of the cell cycle, respectively. the number of ki67-positive and brdu-positive crypt cells was significantly reduced ~40% in gata6Δie mice, as previously reported, but unchanged in spdefko mice, as compared to controls. the number of ki67- and brdu-positive crypt cells in dko mice was also similar to controls, indicating that the additional deletion of spdef rescues the decrease in crypt cell proliferation observed in gata6Δie mice. these data support the notion that gata maintains crypt cell proliferation by down-regulating spdef gene expression. spdef does not regulate the secretory cell differentiation function of gata6 previously, conditional deletion of gata in the ileum resulted in a 25-40% reduction in the number of chromogranin a -positive cells, and in the mrna abundances of chga and neurog, consistent with a decrease in enteroendocrine lineage commitment. though not statistically significant, we found a 15-50% reduction in the number of chga-positive cells, and in the mrna abundances of chga and neurog in the gata6Δie mice, in general agreement with our previous study. we further found that although the pattern for these three measurements in the spdefko mice was similar to that in ctl mice, consistent with previous data for distal intestine, the pattern in the dko mice was similar to that in the gata6Δie mice. together, these data show that enteroendocrine cell commitment is reduced in both gata6Δie and dko mice, indicating that gata promotes enteroendocrine cell commitment independently of spdef. previously, we also showed that conditional gata deletion resulted in a transformation of paneth cells into mucin- -enriched goblet-like cells at the base of crypts. in the present study, immunostaining for crs4c, a paneth-specific marker, was slightly reduced in spdefko mice as compared to controls, consistent with previous data, but was greatly reduced in both gata6Δie and dko mice as compared to controls. the number of crypt cross-sections with at least one crs4c-positive cell was nearly 100% in ctl and spdefko mice, but was less than 30% in both gata6Δie and dko mice. the mrna abundance for lysozyme, another paneth marker, was not significantly different between ctl and spdefko mice, but was significantly reduced in gata6Δie and dko mice as compared to controls. pas staining was greatly increased in crypts of gata6Δie mice, as previously shown, noticeably reduced in spdefko mice, especially in villus goblet cells, as previously shown, and similar to controls in dko mice. muc immunostaining was greatly enriched in crypts of gata6Δie mice, consistent with our previous report, but similar to controls in spdefko and dko mice. generally consistent with this observation, muc mrna abundance in gata6Δie mice was -fold higher as compared to controls, though not statistically significant, whereas muc mrna abundances in spdefko and dko mice were similar to controls. these data indicate that the additional deletion of spdef did not rescue the decrease in the terminal differentiation of paneth cells observed in gata6Δie mice, but did at least partially prevent their conversion to muc2-enriched goblet-like cells. thus, spdef is not necessary for the gata-mediated differentiation of paneth cells, but does function in their default differentiation into goblet-like cells in the absence of gata this is consistent with the general function of spdef to promote the differentiation of goblet cells. in spite of the common secretory cell phenotypes in gata deletion and spdef over-expression models, our data show that spdef deletion did not rescue the gata knockout defects in enteroendocrine lineage commitment or in paneth cell differentiation. closer scrutiny suggests that the secretory cell phenotypes in the gata deletion and spdef over-expression models are not identical. while the decline in enteroendocrine and paneth cells, and accumulation of goblet cells, appear similar, the underlying alteration in paneth and goblet cell differentiation is different. conditional gata deletion has no effect on the normal commitment and differentiation of goblet cells on villi but reveals a conversion of paneth cells at the base of crypts into a goblet-like cell type. these cells do not express defensins, but express abundant muc2; they also express abundant sox and ephb, indicating that they are committed and targeted paneth cells. on the other hand, conditional spdef over-expression results in a generalized increase in goblet cells at the expense of all other epithelial cell types. hence, these mice show a decline in paneth cell specification rather than a defect in the terminal differentiation of committed paneth cells, as observed in our conditional gata knockout model. spdef does not regulate the absorptive enterocyte gene expression function of gata6 we next examined whether spdef loss affected gata6-dependent absorptive enterocyte gene expression. conditional gata deletion resulted in a down-regulation of specific absorptive enterocyte genes in ileum that include lipid transporters and apolipoproteins, and an up-regulation of genes in absorptive enterocytes normally not expressed or expressed at low levels in small intestine, but expressed at high levels in colon. using marker genes for these two patterns, apolipoprotein a and carbonic anyhdrase, respectively, we found that their mrna abundances were down-regulated and up-regulated, respectively, in gata6Δie mice, consistent with our previous findings. apoa and car mrna abundance in spdefko mice was similar to controls, whereas that in dko mice was similar to gata6Δie, indicating that spdef does not play a role in mediating the conditional gata knockout alteration in absorptive enterocyte gene expression. gata is expressed in crypts and in mature absorptive enterocytes, whereas spdef is expressed in secretory progenitors, and in goblet and paneth cells, but is not expressed in mature absorptive enterocytes. while it is possible that spdef could instruct gene expression in absorptive enterocytes through a process that originates in progenitors early in the differentiation process, we found that spdef did not regulate the gata targets studied here. as in the gata4-specific pathway, in which gata regulates its targets in mature absorptive enterocytes on villi rather than in crypt progenitors, we believe that gata also regulates absorptive enterocyte gene expression within mature absorptive enterocytes on villi.  previously, we defined two fundamental pathways of gata regulation in the small intestine, one mediated exclusively by gata, and one regulated by gata or gata. in the gata4-specific pathway, gata but not gata activates and represses a subset of absorptive enterocyte genes, and by virtue of its expression in the proximal 85% of small intestine and lack of expression in distal ileum, distinguishes proximal intestinal from distal ileal gene expression and function. for the gata4/gata6-redundant pathway, either gata or gata promote intestinal epithelial renewal by supporting crypt cell proliferation, enteroendocrine lineage commitment, paneth cell differentiation, and absorptive enterocyte gene expression. here, we show that the maintenance of crypt cell proliferation function of the intestinal gata4/gata6-redundant pathway is dependent on spdef. specifically, our data support the notion that gata4/gata promotes crypt cell proliferation by directly repressing spdef gene expression. gata has been suggested to be oncogenic in multiple cancers and pre-cancerous lesions, including barrett’s esophagus, gastric cancer, pancreatic cancer, and colon cancer. gata expression is up-regulated in colon cancer epithelial cells, as well as in non-malignant cells along the stromal margins in human colorectal cancer, but it should be noted that it has not been determined whether this is a correlative, causative or protective event. recently, noah et al. showed that spdef functions as a colorectal tumor suppressor. in colorectal tumors from patients, loss of spdef was observed in approximately 85% of tumors and correlated with progression from normal tissue, to adenoma, to adenocarcinoma. further, spdef inhibited the expression of β-catenin-target genes in mouse colon tumors, and interacted with β-catenin to block its transcriptional activity in colorectal cancer cell lines, resulting in lower levels of cyclin d and c-myc. pathway analysis showed that gata deletion also modulated c-myc targets and that the gata6/spdef overlapping gene set likely functions in wnt signaling. thus, our data here suggest that the possible tumorigenic effects of gata could be mediated by its repression of spdef gene expression. 
to test this hypothesis, we defined the functions most likely regulated by the overlapping gata6/spdef target gene set in mouse intestine, delineated the relationship between gata chromatin occupancy and spdef gene regulation in caco- cells, and determined the extent to which prevention of spdef up-regulation by spdef knockout rescues the gata phenotype in conditional gata knockout mouse ileum. gata occupies a genetic locus located 40 kb upstream of the spdef transcription start site, consistent with direct regulation of spdef gene expression by gata prevention of spdef up-regulation in conditional gata knockout mouse ileum by the additional deletion of spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression. deletion of intestinal gata and gata results in an altered proliferation/differentiation phenotype, and an up-regulation of sam pointed domain containing ets transcription factor, a transcription factor recently shown to act as a tumor suppressor. in human caco- cells, gata knockdown results in an up-regulation of spdef gene expression, modeling our mouse gata knockout data. the hypothesis to be tested is that intestinal gata4/gata functions through spdef by repressing spdef gene expression. gata transcription factors are essential for self-renewal of the small intestinal epithelium. gata is expressed in the proximal 85% of small intestine while gata is expressed throughout the length of small intestine. the goal of this study is to determine to what extent spdef mediates the downstream functions of gata4/gata in the small intestine. spdef is a key, immediate downstream effecter of the crypt cell proliferation function of gata4/gata in the small intestine.
 gata transcription factors are essential for self-renewal of the small intestinal epithelium. gata is expressed in the proximal 85% of small intestine while gata is expressed throughout the length of small intestine. deletion of intestinal gata and gata results in an altered proliferation/differentiation phenotype, and an up-regulation of sam pointed domain containing ets transcription factor, a transcription factor recently shown to act as a tumor suppressor. the goal of this study is to determine to what extent spdef mediates the downstream functions of gata4/gata in the small intestine. the hypothesis to be tested is that intestinal gata4/gata functions through spdef by repressing spdef gene expression. to test this hypothesis, we defined the functions most likely regulated by the overlapping gata6/spdef target gene set in mouse intestine, delineated the relationship between gata chromatin occupancy and spdef gene regulation in caco- cells, and determined the extent to which prevention of spdef up-regulation by spdef knockout rescues the gata phenotype in conditional gata knockout mouse ileum.  using publicly available profiling data, we found that 83% of gata6-regulated genes are also regulated by spdef, and that proliferation/cancer is the function most likely to be modulated by this overlapping gene set. in human caco- cells, gata knockdown results in an up-regulation of spdef gene expression, modeling our mouse gata knockout data. gata occupies a genetic locus located 40 kb upstream of the spdef transcription start site, consistent with direct regulation of spdef gene expression by gata prevention of spdef up-regulation in conditional gata knockout mouse ileum by the additional deletion of spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression.  spdef is a key, immediate downstream effecter of the crypt cell proliferation function of gata4/gata in the small intestine. gata6spdefcrypt cell proliferationintestinal differentiation 