Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domainswapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbf␤, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

Invertebrate and vertebrate Runt-related proteins are transcription factors involved in the regulation of a variety of celldifferentiation events and aberrant functions of members of this protein family correlate with developmental abnormalities and neoplastic transformations (1-5). In particular, the mouse Runt-related protein, core-binding factor ␣2 (Cbfa2) 1 is essential for fetal liver hematopoiesis (6 -8) and its human homolog, AML1, is frequently targeted by chromosomal translocations that lead to acute myeloid leukemia (4, 6). A related mouse protein, Cbfa1, plays an essential role in osteoblast differentiation and mutations interfering with its function correlate with defects in ossification in mice and humans (3, 5, 9 -11). Studies in invertebrate and vertebrate species implicate Runt-related proteins in both transactivation and transcriptional repression, suggesting that their transcription functions may be regulated in context-dependent ways by interactions with other proteins (1, 9, 12-17). In this regard, Drosophila Runt has recently (18) been shown to interact with the protein Groucho, a general transcriptional repressor involved in a variety of gene regulatory events (19 -21). In particular, genetic studies show that repression of certain Runt-regulated genes is dependent on interaction with Groucho and is sensitive to Groucho dosage (18). These results implicate Groucho in the regulation of the transcriptional functions of Runt in Drosophila.A number of observations have suggested that the functions of mammalian Runt-related proteins are also modulated by Groucho homologs, designated as the transducin-like Enhancer of split (TLE) or Groucho-related gene products 1 through 4 (hereafter referred to as TLE1-4) (22-24). TLE proteins are co-expressed with the Runt-related proteins, Cbfa1 and Cbfa2/ AML1 (AML1), in a variety of cell types (7,14,(25)(26)(27). In addition, AML1 and TLE proteins can physically interact with each other (28). Furthermore, transient transfection studies in mammalian cells have shown that TLE proteins can inhibit the transactivation mediated by both Cbfa1 and AML1 (14,28,29). Together, these findings strongly suggest that TLE proteins are involved in the regulation of the transcriptional functions of mammalian Runt-related proteins.Studies in Drosophila have also implicated a second evolutionarily conserved family of transcription factors in the regulation of the functions of Runt-related proteins. Specifically, the basic helix loop helix proteins of the Drosophila Hairy/ Enhancer of split (HES) family are co-expressed with Groucho and Runt in a variety of cell types and physically interact with Groucho (18,20,21,30). Moreover, genetic studies in Drosophila show that runt and HES genes contribute to common gene regulatory events important for sex determination and segmentation (30 -32). Mammalian HES and runt-related genes are also co-expressed with TLE genes in a variety of cell types and their protein products participate in common developmental mechanisms (14,27,33,34). To...

The neural crest and sensory placodes arise from a region of the embryonic ectoderm that lies between the neural plate and future epidermis. While some of the signalling pathways that are involved in cell fate determination at the border of the neural plate have been characterised, it is still unclear how different signals are integrated. Transcription factors of the DLX gene family that may mediate such cell fate decisions are expressed at the border of the neural plate. Here, we demonstrate that DLX5 is involved in positioning this border by repressing neural properties and simultaneously by promoting the formation of border-like cells that express the neural fold markers MSX1 and BMP4 and the preplacodal region marker SIX4. However, DLX5 is not sufficient to impart epidermal character or to specify cell fates that arise at the border of the neural plate, like neural crest or fully formed sensory placodes, in a cell-autonomous manner. Additional signals are generated when mature neural plate and epidermis interact and these are required for neural crest formation. We propose that patterning of the embryonic ectoderm is a multistep process that sequentially subdivides the ectoderm into regions with defined cell fates.

Hairy/Enhancer of split 1 (Hes1) is a mammalian transcriptional repressor that plays crucial roles in the regulation of several developmental processes, including neuronal differentiation. The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1. It is shown here that Hes1 associates with the nuclear matrix, the ribonucleoprotein network of the nucleus that plays important roles in transcriptional regulation. Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that is also required for transcriptional repression. This domain contains the WRPW motif that acts as a binding site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1. This corepressor role is not modulated by the Groucho-related gene product Grg5. In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with Groucho and Hes1, can inhibit both the Groucho⅐Hes1 interaction and the transcription repression ability of Hes1. Together, these observations suggest that transcriptional repression by Hes1 requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of Groucho⅐Hes1 complexes.