The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.

Physiological Wnt signaling is required for the maintenance of the crypt progenitor phenotype and controls the proliferation/differentiation switch in the adult, self-renewing intestinal epithelium (33). A constitutively active Tcf/␤-catenin transcription complex, resulting from mutations in adenomatous polyposis coli (APC), Axin, or ␤-catenin, is the primary transforming factor in colorectal cancer (CRC) (25,26,32); aberrant Tcf/␤-catenin activity results in a transcriptional profile in CRC cells similar to that which is physiologically driven by Tcf/␤-catenin in the crypt stem/progenitor cells of the intestine (49). Through candidate gene approaches and microarray technology, a large number of genes have been uncovered whose expression levels are altered upon abrogation or activation of the Wnt pathway (for references, see http://www .stanford.edu/ϳrnusse/pathways/targets.html). It remains unclear whether the affected genes are direct or indirect targets of the Tcf/␤-catenin transcription factor complex. cis-regulatory elements directly bound by Tcf have been identified for only a few candidate genes. Such studies have been mostly limited to regulatory regions close to the transcription start site (TSS) of candidate genes (e.g., see reference 17). A comprehensive identification of regulatory elements is essential for a more complete understanding of the transcriptional repertoire driven by the Wnt pathway and the elucidation of the molecular mechanisms by which Tcf and ␤-catenin control the transcription of their target genes.A recent approach taken to achieve such goals is chromatin immunoprecipitation (ChIP)-coupled DNA microarray analysis (ChIP-on-chip), which couples the immunoprecipitation of chromatin-bound transcription factors with the identification of the bound DNA sequences through hybridization on DNA microarrays (35). This approach has been used to generate, among others, a comprehensive map of active, preinitiation complex-bound promoters in human fibroblast cells (24). Microarrays covering the nonrepetitive sequence of chromosomes 21 and 22 have allowed the study of histone H3 methylation and acetylation patterns in human hepatoma cells (5) and estrogen receptor binding sites in breast cancer cells (8). The latter study revealed selective binding of estrogen receptor (ER) to a limited number of sites, most of which were distant from the TSSs of ER-regulated genes (8). Similar conclusions were put forth by work examining the in vivo binding of transcription factors Sp1, c-Myc, and p53 along chromosomes 21 and 22: most binding sites identified do not correspond to the proximal promoters of protein-coding genes but rather lie within or immediately 3Ј to well-characterized genes or are significantly correlated with noncoding RNAs (10). Collectively these studies point to the necessity of interrogating entire genomes for the comprehensive determination of in vivo-occupied binding sites (9,23,52,54).In the present work, we used a combination of ChIP and location analysis with genome-wide tiling arrays...

Cross-regulatory cascades between hepatic transcription factors have been implicated in the determination of the hepatic phenotype. Analysis of recruitments to regulatory regions and the temporal and spatial expression pattern of the main hepatic regulators during liver development revealed a gradual increase in complexity of autoregulatory and cross-regulatory circuits. Within these circuits we identified a core group of six transcription factors, which regulate the expression of each other and the expression of other downstream hepatic regulators. Changes in the promoter occupancy patterns during development included new recruitments, release, and exchange of specific factors. We also identified promoter and developmental stage-specific dual regulatory functions of certain factors as an important feature of the network. Inactivation of HNF-4␣ in embryonic, but not in adult, liver resulted in the diminished expression of most hepatic factors, demonstrating that the stability of the network correlates with its complexity. The results illustrate the remarkable flexibility of a self-sustaining transcription factor network, built up by complex dominant and redundant regulatory motifs in developing hepatocytes.[Keywords: Hepatocyte development; transcription factor; regulatory network] Supplemental material is available at http://www.genesdev.org.