CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, we conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins on chondrocyte proliferation, differentiation, and the expression of chondrocyteassociated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes in vitro, whereas Ccn3 expression drastically increased. In contrast, the addition of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes, whereas decreasing the Ccn3 expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

The yeast KEOPS protein complex comprising Kae1, Bud32, Cgi121, Pcc1 and Gon7 is responsible for the essential tRNA threonylcarbamoyladenosine (t6A) modification. Deletion of genes coding for the KEOPS subunits also affects telomere elongation and transcriptional regulation. In the present work, the crystal structure of Bud32/Cgi121 in complex with ADP revealed that ADP is bound in the catalytic site of Bud32 in a canonical manner characteristic of Protein Kinase A (PKA) family proteins. We found that Gon7 forms a stable heterodimer with Pcc1 and report the crystal structure of the Pcc1-Gon7 heterodimer. Gon7 interacts with the same Pcc1 region engaged in the archaeal Pcc1 homodimer. We further show that yeast KEOPS, unlike its archaeal counterpart, exists as a heteropentamer in which Gon7, Pcc1, Kae1, Bud32 and Cgi121 also adopt a linear arrangement. We constructed a model of yeast KEOPS that provides structural insight into the role of Gon7. The model also revealed the presence of a highly positively charged crater surrounding the entrance of Kae1 that likely binds tRNA.