A new chromatin structure at the eukaryotic centromere
At the eukaryotic centromere, more than 100 proteins participate to build a kinetochore: microtubule-capturing machinery. How the kinetochore is built at the centromere remained elusive. Here, we identified a novel kinetochore complex consisting of CENP-T, -W, -S, -X that form a histone-like heterotetramer and wrap DNA similar to canonical histones. Our results extend the current 'histone-code' hypothesis utilizing histone modifications and histone variants to 'chromatin-code' including histone-fold protein complexes.
HFSP Career Development Award holder Tatsuya Nishino and Young Investigator Grant holder Iain Cheeseman and colleaguesauthored on Fri, 16 March 2012
During mitosis, replicated sister chromatids are segregated faithfully into two daughter cells. On each chromosome, the ‘Centromere’ is a region where more than 100 proteins assemble to form a platform for equal chromosome segregation. This platform is called a ‘Kinetochore’ where microtubules emanating from two daughter centrosomes attach to the chromosome and form a bipolar spindle. The Centromere is specified epigenetically but how the kinetochore on the centromere is organized to form a microtubule capturing machinery remains a mystery. As the kinetochore is formed on centromeric DNA, it is likely that the basal part of the kinetochore makes a strong contact with DNA. We have previously reported the CENP-T-W complex as a DNA binding protein among kinetochore components (Hori et al., 2008). More recently, we demonstrated that targeting CENP-T to a non-centromeric region induced an artificial kinetochore (Gascoigne et al., 2011), suggesting that the CENP-T-W complex forms a platform for other kinetochore components. However, it has been unclear how CENP-T-W is targeted to the centromere and how its DNA binding drives kinetochore assembly.
Figure: Ribbon diagram of CENP-T-W-S-X tetramer(left), CENP-S-X tetramer(middle), and the nucleosome(right). The structure is viewed from the dyad axis of the tetrameric complex. In all cases, a four-helix bundle stabilizes the complex. We assume CENP-T-W-S-X tetramer and CENP-S-X tetramer use a similar interface with the Histone H3-H4 complex to wrap DNA around itself.
To gain insight into the CENP-T-W complex function, we analyzed its structure. We determined the crystal structure of the CENP-T-W DNA binding domain. The crystal structure revealed that CENP-T-W forms a dimer. Overall fold was similar to canonical histones. Based on similarity, we designed mutations on possible DNA binding surfaces. These mutant proteins showed reduced DNA binding activity and CENP-T-deficient chicken DT40 cells expressing these mutant proteins did not form a functional kinetochore. Thus, we conclude that the DNA binding activity of the CENP-T-W complex is essential for kinetochore assembly. Previously, we identified a second histone-fold complex consisting of CENP-S and CENP-X which are stable kinetochore components (Amano et al., 2009). To understand its function, we determined the crystal structure of the CENP-S-X complex. CENP-S-X was also similar to histone but it formed a tetramer. Mutations in the putative DNA binding region of CENP-S-X revealed that it was also important for kinetochore assembly.
To our surprise, comparison of the CENP-S-X and CENP-T-W structures revealed that the overall structure was very similar. The similarity is further notable at the oligomerization region within CENP-S and CENP-T. This suggested that these two complexes may form a higher order complex. When the two proteins were mixed together, CENP-S-X and CENP-T-W formed a CENP-T-W-S-X heterotetramer. By X-ray crystallography, we confirmed that CENP-T-W-S-X forms a heterotetramer. Mutations in the tetramer interface inhibited complex formation and cells expressing these mutant proteins did not form a functional kinetochore. In vitro DNA binding analysis revealed that CENP-T-W-S-X heterotetramer forms a distinct protein-DNA complex that is different from individual CENP-T-W or CENP-S-X. The CENP-T-W-S-X heterotetramer induced supercoils into DNA like canonical histones and the activity was abolished using mutant proteins that are defective for DNA binding, suggesting that the heterotetramer wraps DNA similar to canonical histones.
In conclusion, our structural, biochemical and cell biological analysis suggest that CENP-T-W-S-X forms a unique centromeric chromatin and plays an essential role in the formation of functional kinetochore.
CENP-T-W-S-X Forms a Unique Centromeric Chromatin Structure with a Histone-like Fold. Nishino T, Takeuchi K, Gascoigne KE, Suzuki A, Hori T, Oyama T, Morikawa K, Cheeseman IM, Fukagawa T. Cell. 2012 Feb 3;148(3):487-501.
Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.Gascoigne KE, Takeuchi K, Suzuki A, Hori T, Fukagawa T, Cheeseman IM.Cell. 2011 Apr 29;145(3):410-22.
CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Hori T, Amano M, Suzuki A, Backer CB, Welburn JP, Dong Y, McEwen BF, Shang WH Suzuki E, Okawa K, Cheeseman IM, Fukagawa T. Cell. 2008 Dec 12;135(6):1039-52.
The CENP-S complex is essential for the stable assembly of outer kinetochore structure. Amano M, Suzuki A, Hori T, Backer C, Okawa K, Cheeseman IM, Fukagawa T. J Cell Biol. 2009 Jul 27;186(2):173-82.