Transforming growth factor-b (TGF-b) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-b/ Smad3 inhibits Runx2 function has not been characterized. We show that TGF-b induces histone deacetylation, primarily of histone H4, at the osteocalcin promoter, which is repressed by TGF-b, and that histone deacetylation is required for repression of Runx2 by TGF-b. This repression occurs through the action of the class IIa histone deacetylases (HDAC)4 and 5, which are recruited through interaction with Smad3 to the Smad3/Runx2 complex at the Runx2-binding DNA sequence. Accordingly, HDAC4 or 5 is required for efficient TGF-b-mediated inhibition of Runx2 function and is involved in osteoblast differentiation. Our results indicate that class IIa HDACs act as corepressors for TGF-b/Smad3-mediated transcriptional repression of Runx2 function in differentiating osteoblasts and are cell-intrinsic regulators of osteoblast differentiation.

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mRNA synthesis requires pol 1 II and a set of general transcription factors (GTFs) including TFIIA, TFIIB, TFIID, TFIIF, and TFIIH. The regulation of this process requires a number of coactivator complexes involved in chromatin remodeling and recruitment of transcriptional machinery to the promoter (1-5). In particular, the yeast Mediator complex is required for diverse aspects of transcriptional regulation, including activated transcription and transcriptional repression (7-9). In addition, Mediator improves the efficiency of basal transcription and the phosphorylation of the C-terminal domain (CTD) of the largest subunit of pol II (Rpb1) (6). To accommodate these diverse activities, the Mediator complex is composed of more than 20 polypeptides, including Srb subunits (Srb2, -4, -5, -6, and -7), Med subunits

Transforming growth factor b (TGF-b) inhibits myogenesis and associated gene expression. We previously reported that the TGF-b signaling effector Smad3 mediates this inhibition, by interfering with the assembly of myogenic bHLH transcription factor heterodimers on E-box sequences in the regulatory regions of muscle-specific genes. We now show that TGF-b-activated Smad3 suppresses the function of MEF2, a second class of essential myogenic factors. TGF-b signaling through Smad3 represses myogenin expression independently of E-boxes, and prevents a tethered MyoD-E47 dimer to activate transcription indirectly through MEF2-binding sites. In addition, Smad3 interacts with MEF2C, which requires its MADS domain, and disrupts its association with the SRCfamily coactivator GRIP-1, thus diminishing the transcription activity of MEF2C. Consistent with this physical displacement, TGF-b signaling blocks the GRIP-1-induced redistribution of MEF2C to discrete nuclear subdomains in 10T1/2 cells, and the recruitment of GRIP-1 to the myogenin promoter in differentiating myoblasts. These findings indicate that the TGF-b/Smad3 pathway targets two critical components of the myogenic transcription machinery to inhibit terminal differentiation.

Post-translational sumoylation, the covalent attachment of a small ubiquitin-like modifier (SUMO), regulates the functions of proteins engaged in diverse processes. Often associated with nuclear and perinuclear proteins, such as transcription factors, it is not known whether SUMO can conjugate to cell-surface receptors for growth factors to regulate their functions. Here we show that the type I transforming growth factor-beta (TGF-beta) receptor, T beta RI, is sumoylated in response to TGF-beta and that its sumoylation requires the kinase activities of both T beta RI and the type II TGF-beta receptor, T beta RII. Sumoylation of T beta RI enhances receptor function by facilitating the recruitment and phosphorylation of Smad3, consequently regulating TGF-beta-induced transcription and growth inhibition. T beta RI sumoylation modulates the dissemination of transformed cells in a mouse model of T beta RI-stimulated metastasis. T beta RI sumoylation therefore controls responsiveness to TGF-beta, with implications for tumour progression. Sumoylation of cell-surface receptors may regulate other growth factor responses.

Recent progress in the design and selection of novel zinc finger proteins with desired DNA binding specificities now allows construction of tailor-made DNA-binding proteins that specifically recognize almost any predetermined DNA sequence. Such novel or "designer" zinc finger proteins with desired DNA binding specificities can serve as efficient transcription factors in various mammalian cell lines. In addition, they may be broadly useful in the regulation of endogenous genes in transgenic organisms and eventually in gene therapy applications. In this report, we use a series of transient and stable transfection experiments to demonstrate that the expression of a target gene can be controlled by changing the in vivo concentration of designer zinc finger proteins in a dose-dependent manner. We also report that designer zinc finger proteins can access their binding sites integrated into the genome and function as potent transcription factors. Our results suggest that designer zinc finger transcription factors that specifically recognize appropriate sites in the promoter of a target gene may have broad applications in the postgenomic era.

The yeast Mediator is composed of two subcomplexes, Rgr1 and Srb4, known to be required for diverse aspects of transcriptional regulation; however, their structural and functional organizations have not yet been deciphered in detail. Biochemical analyses designed to determine the subunit composition of the Rgr1 subcomplex revealed that the regulator-interacting subcomplex has a modular structure and is composed of the Gal11, Med9/Cse2, and Med10/Nut2 modules. Genome-wide gene expression and Northern analyses performed in the presence or absence of the various Mediator modules revealed a distinct requirement for the Gal11, Med9/Cse2, and Med10/Nut2 modules in transcriptional repression as well as activation. GST pull-down analysis revealed that the transcriptional repressor Tup1 binds to distinct but overlapping regions of the Gal11 module that were shown previously to be transcriptional activator binding sites. These data suggest that competition between transcriptional activators and repressors for a common binding site in the Mediator and distinct conformational changes in the Mediator induced by repressor binding may underlie the mechanism of transcriptional repression in eukaryotes.

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