A dynamic expression survey identifies transcription factors relevant in mouse digestive tract development.

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.

Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF).

The vertebrate intestine is a model for investigating inductive cellular interactions and the roles of epithelial stem cells in tissue regeneration, and for understanding parallels between development and cancer. We have used serial analysis of gene expression to measure transcript levels across stages in mouse intestine development. The data (http://genome.dfci.harvard.edu/GutSAGE) identify novel differentiation products, potential effectors of epithelial-mesenchymal interactions, and candidate markers and regulators of intestinal epithelium. Transcripts that decline significantly during intestine development frequently are absent from the adult gut. We show that a significant proportion of such genes may be reactivated in human colon cancers. As an example, hepatoma-derived growth factor (HDGF) mRNA is expressed prominently in early gut tissue, with substantially reduced levels after villous epithelial differentiation. HDGF expression is dramatically increased in human colorectal cancers, especially in tumors proficient in DNA mismatch repair, and thus represents a novel marker for a distinctive tumor subtype. HDGF overexpression in fetal intestine explants inhibits maturation, suggesting a role in epithelial differentiation. To investigate the molecular basis for HDGF functions, we isolated components of a nuclear HDGF complex, including heterogeneous nuclear ribonucleoproteins implicated in processing RNA. These genes are regulated in tandem with HDGF during intestine development and one factor, TLS/Fus, is commonly overexpressed in colon cancers. Tumor expression of fetal genes may underlie similarities between developing and malignant tissues, such as self-renewal, invasion and angiogenesis. Our findings also advance understanding of HDGF functions and implicate this developmentally regulated gene in RNA metabolic pathways that may influence malignant behaviors in colorectal cancer.

Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex.

Key molecular lesions in colorectal and other cancers cause beta-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and beta-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/beta-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of beta-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/beta-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction.

Characterization of a novel mammalian Groucho isoform and its role in transcriptional regulation.

The Wnt/beta-catenin/Tcf pathway serves important functions in embryonic development and is constitutively activated in human colorectal cancer. The nuclear output of Wnt signaling is mediated by a complex between DNA-binding proteins of the TCF family and the transcriptional coactivator beta-catenin. Groucho proteins act to repress transcriptional activation by beta-catenin-Tcf complexes, probably by interacting directly with Tcf transcription factors. We have identified several splice forms of the mouse Groucho Grg1 gene expressed in the developing intestine. Prominent among these is a novel and abundant isoform, Grg1-S, which we characterize in this report. Grg1-S has highest homology with the TLE family of large Groucho proteins but features only the amino-terminal Q and glycine- and proline-rich domains typical of the Groucho/AES subfamily. Grg1-S is expressed in development and in several adult mouse tissues. Expression in the adult small intestine is highest at the base of the crypts of Lieberkuhn. Grg1-S acts to antagonize beta-catenin activity in Xenopus axis duplication and luciferase reporter assays in mammalian cells. Taken together, these findings suggest that Grg1-S may operate in conjunction with beta-catenin and Tcf factors to regulate vertebrate gut epithelial cell differentiation.