SummaryChromosome segregation in metazoans requires the alignment of sister-kinetochores onto the metaphase plate. During chromosome alignment, bioriented kinetochores move chromosomes by regulating the plus-end dynamics of the attached microtubules. The bundles of kinetochore-bound microtubules alternate between growth and shrinkage, leading to regular oscillations along the spindle axis. However, the molecular mechanisms that coordinate microtubule plus-end dynamics remain unknown. Here we show that CENP-H, a subunit of the CENP-A NAC/CAD kinetochore complex, is essential for this coordination, as kinetochores lacking CENP-H establish bioriented attachments, but fail to generate regular oscillations, due to an uncontrolled rate of microtubule plus-end turnover. These alterations lead to rapid erratic movements that disrupt metaphase plate organization. Moreover, we show that the abundance of the CENP-A NAC/CAD subunits CENP-H and CENP-I dynamically change on individual sister-kinetochores in vivo, as they preferentially bind the sister-kinetochore attached to growing microtubules, and that one other subunit, CENP-Q, binds microtubules in vitro. Thus, we propose that CENP-A NAC/CAD is a direct regulator of kinetochore-microtubule dynamics, which physically links centromeric DNA to microtubule plusends.

The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome containing a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochemical and biophysical methods and show that it forms a stable octamer containing two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those observed in vivo demonstrating that the octameric structure is physiologically relevant.

In vivo cells receive simultaneous signals from multiple extracellular ligands and must integrate and interpret them to respond appropriately. Here we investigate the interplay between pathways downstream of two transforming growth factor ␤ (TGF-␤) superfamily members, bone morphogenetic protein (BMP) and TGF-␤. We show that in multiple cell lines, TGF-␤ potently inhibits BMP-induced transcription at the level of both BMP-responsive reporter genes and endogenous BMP target genes. This inhibitory effect requires the TGF-␤ type I receptor ALK5 and is independent of new protein synthesis. Strikingly, we show that Smad3 is required for TGF-␤'s inhibitory effects, whereas Smad2 is not. We go on to demonstrate that TGF-␤ induces the formation of complexes comprising phosphorylated Smad1/5 and Smad3, which bind to BMP-responsive elements in vitro and in vivo and mediate TGF-␤-induced transcriptional repression. Furthermore, loss of Smad3 confers on TGF-␤ the ability to induce transcription via BMP-responsive elements. Our results therefore suggest that not only is Smad3 important for mediating TGF-␤'s inhibitory effects on BMP signaling but it also plays a critical role in restricting the transcriptional output in response to TGF-␤.