SUMOylation of the corepressor N-CoR modulates its capacity to repress transcription.

In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein-protein interactions of N-CoR are known, of which mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. On the other hand, the role of posttranslational modifications in corepressor function is not well established. Here, we show that N-CoR is modified by Sumo-1. We found SUMO-E2-conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 as novel N-CoR interaction partners. The SANT1 domain of N-CoR was found to mediate this interaction. We show that K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and that mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, we investigated a possible correlation between the functions of the repression domains and SUMOylation. Coexpression of Ubc9 protein resulted in enhanced N-CoR-dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduced repression of a reporter gene, but also abolished the effect of Ubc9 on transcriptional repression.

An extracellular matrix-specific microarray allowed the identification of target genes downstream of discoidin domain receptors.

The two discoidin domain receptors, DDR1 and DDR2, are tyrosine kinases that are activated by collagen and are essential regulators of cell-matrix communication. However, the target genes downstream of activated DDRs and their physiological significance are largely unknown. Here, we describe a novel method to dissect signaling pathways induced by extracellular matrix (ECM) receptors. Using the doxycycline-inducible repression system (tet-off), we generated human fibrosarcoma and mouse fibroblast cell lines over-expressing DDR1 or DDR2. These cell lines were employed for gene expression analysis using microarrays specific for human and mouse genes coding for ECM proteins or ECM-interacting factors. We found that approximately 10% of the genes studied were up- or down-regulated more than twofold in response to signals generated by over-expressing DDRs. A common event downstream of DDR1 and DDR2 in human and mouse cells was the up-regulation of P-selectin glycoprotein ligand. Key target genes repressed upon DDR activation were agrin, syndecan-1 and alpha3 integrin. ECM-specific microarrays were found a valuable tool to dissect gene expression changes induced by collagen-receptor signaling pathways.