Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2'-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2'-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2'-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells.

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.

Sonic hedgehog (Shh) plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, we developed an in vitro assay for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, we showed that attraction by Shh does not require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons.

SUMMARY Hedgehog (Hh) proteins regulate important developmental processes including cell proliferation and differentiation. While Patched acts as the main Hh receptor, in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, a Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules Boc, Cdon, and Gas1 have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand which binds Patched1 but neither Boc, Cdon, nor Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.

contributed equally to this workThe myocyte enhancer factor-2 (MEF2) proteins are MADS-box transcription factors that are essential for differentiation of all muscle lineages but their mechanisms of action remain largely unde®ned. In mammals, the earliest site of MEF2 expression is the heart where the MEF2C isoform is detectable as early as embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers including atrial natriuretic factor (ANF). However, most of these promoters contain no or low af®nity MEF2 binding sites and they are not signi®cantly activated by any MEF2 proteins in heterologous cells suggesting a dependence on a cardiac-enriched cofactor for MEF2 action. We provide evidence that MEF2 proteins are recruited to target promoters by the cellspeci®c GATA transcription factors, and that MEF2 potentiates the transcriptional activity of this family of tissue-restricted zinc ®nger proteins. Functional MEF2/GATA-4 synergy involves physical interaction between the MEF2 DNA-binding domain and the carboxy zinc ®nger of GATA-4 and requires the activation domains of both proteins. However, neither MEF2 binding sites nor MEF2 DNA binding capacity are required for transcriptional synergy. The results unravel a novel pathway for transcriptional regulation by MEF2 and provide a molecular paradigm for elucidating the mechanisms of action of MEF2 in muscle and non-muscle cells.