Groucho is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila. We are investigating the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducinlike Enhancer of split (TLE) proteins. We report that Groucho/TLEs are associated with chromatin in live cells and that they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones. This interaction is mediated by the H3 aminoterminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing. We also demonstrate that Groucho/ TLEs form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3 and that these interactions may be propagated along the chromosome due to the ability of Groucho/ TLEs to participate in higher order structures.In both invertebrates and vertebrates, signaling mechanisms mediated by the cell-surface receptor Notch control the differentiation of a variety of cell types both by restricting the competence of precursor cells to respond to specific differentiation cues and by providing inductive signals (reviewed in Refs. 1 and 2). Our understanding of the molecular mechanisms underlying the restrictive functions of the Notch pathway points to an important role for proteins involved in transcriptional repression. In particular, molecular genetic analysis of Notch signaling during Drosophila neural development shows that the basic helix-loop-helix (bHLH) 1 proteins encoded by the Enhancer of split complex act at the end point of the Notch cascade by repressing the expression of genes that promote the neural fate (proneural genes) (3-8). This transcriptional function involves the activity of the product of another locus, referred to as groucho (3, 9 -12). This gene was originally implicated in Notch signaling by genetic analysis showing phenotypic interactions between alleles of groucho and other genes involved in the regulation of proneural gene expression (1-3, 13). Molecular studies subsequently showed that the bHLH proteins encoded by the Enhancer of split complex interact with the Groucho protein (10, 12). Groucho also interacts with other bHLH factors related to Enhancer of split proteins; these include the product of the pair rule gene hairy and will be hereafter collectively referred to as Hairy and Enhancer of split-like (HES) proteins (2, 4 -6, 10 -12). It is thought that HES factors recruit the broadly expressed Groucho protein, which lacks DNA-binding ability, to specific DNA sites. Once targeted to DNA, Groucho can repress both basal and activated transcription (12). This particular observation and the demonstration that groucho mutations affect the expression of a variety of ge...

The Drosophila protein Groucho is involved in the regulation of cell-determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin-like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up-regulated and TLE2 and TLE4 are down-regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up-regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non-redundant roles for individual TLE proteins during both cell-determination and cell-differentiation events.

Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family are involved in a variety of cell differentiation mechanisms in both invertebrates and vertebrates. They become recruited to specific promoter regions by forming complexes with a number of different DNA-binding proteins thereby contributing to the regulation of multiple genes. To understand how the functions of Gro/TLE proteins are regulated, it was asked whether their ability to mediate transcriptional repression might be controlled by cell cycle-dependent phosphorylation events. It is shown here that activation of p34 cdc2 kinase (cdc2) with okadaic acid is correlated with hyperphosphorylation of Gro/TLEs. Moreover, pharmacological inhibition of cdc2 activity results in Gro/TLE dephosphorylation. In agreement with these findings, a purified cdc2-cyclin B complex can directly phosphorylate Gro/TLEs in vitro. Two separate Gro/TLE domains, the CcN and SP regions, contain sequences that are phosphorylated by cdc2. Deletion of these sequences is correlated with loss of Gro/ TLE phosphorylation by cdc2 in vitro and okadaic acidinduced Gro/TLE hyperphosphorylation in vivo. In addition, Gro/TLEs are phosphorylated during the G 2 /M phase of the cell cycle, and this is correlated with a decreased nuclear interaction. Finally, the transcription repression ability of Gro/TLEs is enhanced by pharmacological inhibition of cdc2. Taken together, these results demonstrate that Gro/TLE proteins are phosphorylated as a function of the cell cycle and implicate phosphorylation events occurring during mitosis in the negative regulation of Gro/TLE activity.In both invertebrates and vertebrates, transcriptional corepressor proteins of the Groucho/transducin-like Enhancer of split (Gro/TLE) 1 family play crucial roles in the regulation of a variety of cell differentiation mechanisms. In particular, Drosophila Gro is required for sex determination, segmentation, and neural development (1). Vertebrate Gro/TLE proteins contribute to the regulation of neuronal development (2), patterning of the neural tube (3, 4), skeletogenesis (5, 6), hematopoiesis (7-9), and myogenesis (10, 11). Gro/TLE proteins have no intrinsic DNA binding activity but can be targeted to specific gene regulatory regions due to their ability to interact with a number of different DNA-binding transcription factors. Examples of these include basic helixloop-helix proteins of the Hes family (12-17), Runt homology domain proteins of the Runt/RUNX family (7,18,19), homeodomain factors containing engrailed homology region 1 motifs (3, 20 -24), winged-helix domain transcription factors (23, 25), and high mobility group box proteins (7, 9). By virtue of these multiple interactions, the general transcription repression activity of Gro/TLE proteins can be recruited in context-dependent manners to specific target genes.Transcriptional repression by Gro/TLE family members is thought to be mediated by at least two mechanisms. Oligomeric Gro/TLEs can interact with both histones (26, 27) ...