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1.
Physiol Genomics ; 42(1): 115-25, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20354101

ABSTRACT

Ablation of the mouse genes for Onecut-2 and Onecut-3 was reported previously, but characterization of the resulting knockout mice was focused on in utero development, principally embryonic development of liver and pancreas. Here we examined postnatal development of these Onecut knockout mice, especially the critical period before weaning. Onecut-3 knockout mice develop normally during this period. However, Onecut-2 knockout mice fail to thrive, lagging behind their littermates in size and weight. By postnatal day (d)19, they are consistently 25-30% smaller. Onecut-2 knockout mice also have a much higher level of mortality before weaning, with only approximately 70% survival. Interestingly, Onecut-2 knockout mice that are heterozygous for the Onecut-3 knockout allele are diminished even further in their ability to thrive. They are approximately 50-60% as large as their normal-sized littermates at d19, and less than half of these mice survive to weaning. As reported previously, the Onecut-2/Onecut-3 double knockout is a perinatal lethal. Microarray technology was used to determine the effect of Onecut-2 ablation on gene expression in duodenum, whose epithelium has among the highest levels of Onecut-2. A subset of intestinally expressed genes showed dramatically altered patterns of expression. Many of these genes encode proteins associated with the epithelial membrane, including many involved in transport and metabolism. Previously, we reported that Onecut-2 was critical to temporal regulation of the adenosine deaminase gene in duodenum. Many of the genes with altered patterns of expression in Onecut-2 knockout mouse duodenum displayed changes in the timing of gene expression.


Subject(s)
Failure to Thrive/genetics , Gene Expression Profiling , Homeodomain Proteins/genetics , Intestine, Small/metabolism , Transcription Factors/genetics , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Animals, Newborn , Body Size/genetics , Body Size/physiology , Body Weight/genetics , Body Weight/physiology , Cluster Analysis , Duodenum/growth & development , Duodenum/metabolism , Failure to Thrive/metabolism , Female , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Immunohistochemistry , Intestine, Small/growth & development , Male , Mice , Mice, Inbred Strains , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription Factors/metabolism
2.
J Biol Chem ; 281(43): 32263-71, 2006 Oct 27.
Article in English | MEDLINE | ID: mdl-16950765

ABSTRACT

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.


Subject(s)
Enhancer Elements, Genetic , Intestinal Mucosa/physiology , Onecut Transcription Factors/metabolism , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Adult , Animals , Animals, Newborn , Base Sequence , Binding, Competitive , Chromosome Mapping , DNA Footprinting , Deoxyribonuclease I/metabolism , Duodenum/enzymology , Duodenum/physiology , Gene Expression Regulation, Enzymologic , Humans , Intestinal Mucosa/enzymology , Mice , Mice, Transgenic , Molecular Sequence Data , Mutagenesis , Onecut Transcription Factors/chemistry , Onecut Transcription Factors/genetics , Plasmids , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transgenes
3.
J Mol Histol ; 36(1-2): 15-24, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15703995

ABSTRACT

The ability of the GATA family of factors to interact with numerous other factors, co-factors, and repressors suggests that they may play key roles in tissues and cells where they are expressed. Adult mouse small intestine has been shown to express GATA-4, GATA-5, and GATA-6, where they have been implicated in the activation of a number of intestinal genes. Determination of which GATA factor(s) are involved in a specific function in tissues expressing multiple family members has proven difficult. The immunohistochemical analysis presented here demonstrate that within the mouse small intestine GATA-4/-5/-6 are found to be uniquely distributed among the various differentiated lineages of the intestinal epithelium. Among differentiated cells GATA-4 is found only in the villous enterocytes. GATA-5 is absent from enterocytes, but was found in the remaining lineages: goblet, Paneth and enteroendocrine. Additionally, high levels of GATA-6 are found in only one of these differentiated cell types, the enteroendocrine lineage. The observed distribution suggests that the GATA factors may have distinct roles in lineage allocation, lineage maintenance, and/or terminal differentiation events in small intestine.


Subject(s)
DNA-Binding Proteins/analysis , Intestinal Mucosa/cytology , Intestine, Small/cytology , Transcription Factors/analysis , Animals , Cell Differentiation , Cell Lineage , DNA-Binding Proteins/metabolism , GATA4 Transcription Factor , GATA5 Transcription Factor , GATA6 Transcription Factor , Intestinal Mucosa/chemistry , Intestinal Mucosa/metabolism , Mice , Transcription Factors/metabolism
4.
J Biol Chem ; 280(13): 13195-202, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15677472

ABSTRACT

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.


Subject(s)
Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Duodenum/growth & development , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Animals, Newborn , Base Sequence , Binding Sites , CDX2 Transcription Factor , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Erythroid-Specific DNA-Binding Factors , GATA4 Transcription Factor , Genes, Reporter , Humans , Mice , Mice, Transgenic , Models, Genetic , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Neurofibromin 1/metabolism , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Nucleic Acid , Time Factors , Trans-Activators/metabolism , Transcription, Genetic , Transgenes , YY1 Transcription Factor
5.
Am J Physiol Gastrointest Liver Physiol ; 284(6): G1053-65, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12571085

ABSTRACT

The purine metabolic gene adenosine deaminase (ADA) is expressed at high levels in a well-defined spatiotemporal pattern in the villous epithelium of proximal small intestine. A duodenum-specific enhancer module responsible for this expression pattern has been identified in the second intron of the human ADA gene. It has previously been shown that binding of the factor PDX-1 is essential for function of this enhancer. The studies presented here examine the proposed roles of GATA factors in the enhancer. Site-directed mutagenesis of the enhancer's GATA binding sites crippled enhancer function in 10 lines of transgenic mice, with 9 of the lines demonstrating <1% of normal activity. Detailed studies along the longitudinal axis of mouse small intestine indicate that GATA-4 and GATA-5 mRNA levels display a reciprocal pattern, with low levels of GATA-6 throughout. Interestingly, gel shift studies with duodenal nuclear extracts showed binding only by GATA-4.


Subject(s)
Adenosine Deaminase/genetics , DNA-Binding Proteins/metabolism , DNA/metabolism , Duodenum/metabolism , Enhancer Elements, Genetic/genetics , Response Elements/genetics , Transcription Factors/metabolism , Transcriptional Activation/genetics , Animals , Animals, Genetically Modified , Base Sequence , Binding Sites , DNA/genetics , Duodenum/enzymology , GATA4 Transcription Factor , Intestinal Mucosa/enzymology , Intestinal Mucosa/metabolism , Mice , Molecular Sequence Data , Mutation , Organ Specificity , Protein Binding
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