Tissue-restricted transcription factors (TFs), which confer specialized cellular properties, are usually identified through sequence homology or cis-element analysis of lineage-specific genes; conventional modes of mRNA profiling often fail to report non-abundant TF transcripts. We evaluated the dynamic expression during mouse gut organogenesis of 1381 transcripts,covering nearly every known and predicted TF, and documented the expression of approximately 1000 TF genes in gastrointestinal development. Despite distinctive structures and functions, the stomach and intestine exhibit limited differences in TF genes. Among differentially expressed transcripts, a few are virtually restricted to the digestive tract, including Nr2e3,previously regarded as a photoreceptor-specific product. TFs that are enriched in digestive organs commonly serve essential tissue-specific functions, hence justifying a search for other tissue-restricted TFs. Computational data mining and experimental investigation focused interest on a novel homeobox TF, Isx,which appears selectively in gut epithelium and mirrors expression of the intestinal TF Cdx2. Isx-deficient mice carry a specific defect in intestinal gene expression: dysregulation of the high density lipoprotein (HDL) receptor and cholesterol transporter scavenger receptor class B, type I (Scarb1). Thus,integration of developmental gene expression with biological assessment, as described here for TFs, represents a powerful tool to investigate control of tissue differentiation.
We have identified a cDNA for pleckstrin 2 that is 39% identical and 65% homologous to the original pleckstrin. Like the original pleckstrin 1, this protein contains a pleckstrin homology (PH) domain at each end of the molecule as well as a DEP (Dishevelled, Egl-10, and pleckstrin) domain in the intervening sequence. A Northern blot probed with the full-length cDNA reveals that this homolog is ubiquitously expressed and is most abundant in the thymus, large bowel, small bowel, stomach, and prostate. Unlike pleckstrin 1, this newly discovered protein does not contain obvious sites of PKC phosphorylation, and in transfected Cos-7 cells, it is a poor substrate for phosphorylation, even after PMA stimulation. Cells expressing pleckstrin 2 undergo a dramatic shape change associated with actin rearrangement, including a loss of central F-actin and a redistribution of actin toward the cell cortex. Overexpression of pleckstrin 2 causes large lamellipodia and peripheral ruffle formation. A variant of pleckstrin 2 lacking both PH domains still had some membrane binding but did not efficiently induce lamellipodia, suggesting that the PH domains of pleckstrin 2 contribute to lamellipodia formation. This work describes a novel, widely expressed, membrane-associating protein and suggests that pleckstrin 2 may help orchestrate cytoskeletal arrangement.Pleckstrin homology, or PH, 1 domains are amino acid motifs that are capable of binding polyphosphoinositides and regulating protein function (1-6). Frequently, the binding of polyphosphoinositides to the PH domains within a protein localizes the molecules to the cell membrane (7). In addition, some PH domains may interact with other targets such as the /␥ subunits of heterotrimeric G proteins (8 -11) or protein kinase C (12-14). The structure of several PH domains complexed to inositol trisphosphate has been solved (15, 16), confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain.Pleckstrin 1 is a 40-kDa protein containing the prototypic PH domains at its amino and carboxyl termini. It was first described as a major substrate for protein kinase C (PKC) in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Although its function in vivo is unclear, heterologously expressed pleckstrin can affect second messenger-based signaling events mediated by phospholipase C, PI3K␥, and 5Ј-inositol phosphatases (17-19). Overexpression and microinjection studies suggest that pleckstrin 1 is membrane-localized, induces a shift of F-actin toward the cell cortex, and participates in the production of lamellipodia (3). These functions are tightly regulated by PKC-mediated phosphorylation of three residues (Ser 113 , Thr 114 , and Ser 117 ) located near, but not within, the amino-terminal PH domain (24). Recently, a DEP (Dishevelled, Egl-10, and pleckstrin) domain has been described in pleckstrin 1, but the function of this motif is unknown (20).Regulation of pleckstrin 1 is uni...
Pathways relevant to cancer are well known to overlap with fetal development, as reflected in reactivation of embryonic genes in tumors. However, molecular evidence for this notion has gathered in piecemeal fashion, and systematic approaches have rarely been applied to gauge the extent and global characteristics of the overlap in gene expression between developing tissues and cancer. The fraction of genes that is expressed aberrantly in a given cancer and also developmental in primary function is unknown, and the tissue specificity of recapitulated gene expression remains unexplored. We developed a statistical method to relate expression profiles from human colon cancer and diverse nonintestinal tumors to transcripts that decline in expression with epithelial differentiation in the fetal mouse gut. For genes that are overexpressed in colon cancer, we computed 8% to 19% likelihood that they were expressed transiently during epithelial morphogenesis in intestine development. Among genes dysregulated in other tumors, the corresponding likelihood fell between 1% and 6%. Similarly, low probabilities were obtained when we compared genes not overexpressed in colon cancer with transcriptional profiles in intestine organogenesis. Genes that increase after fetal gut epithelial differentiation were not differentially represented between cancerous and normal colon. Our findings systematically characterize the global extent and tissue specificity of developmental expression programs in colorectal cancer and illustrate the use of such an approach to identify candidate biomarkers and therapeutic targets. (Cancer Res 2005; 65(19): 8715-22)
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