CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

Abstract

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromereassociated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPRmediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a 2 conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore. IntroductionThe centromere is an essential chromosomal region which ensures equal partitioning of chromosomal DNA during cell division [1]. In all eukaryotes, faithful chromosome segregation requires each chromosome to interact accurately with microtubule fibers from the mitotic or meiotic spindle. This interaction is mediated by the kinetochore, a macromolecular protein complex that assembles on centromeric DNA [2]. The centromere-proximal inner kinetochore hosts components of the Constitutive Centromere Associated Network (CCAN), a group of up to 16 different proteins present throughout the cell cycle that create the centromere-kinetochore interface. Upon cell division, the CCAN recruits the centromere distal outer kinetochore complex, which is composed of the KMN network (Knl1, the Mis12 and the Ndc80 complex) [3]. This recruitment is enabled by two CCAN subunits, CENP-C and CENP-T, that physically interact with subunits of the Mis12 and Ndc80 complexes [4][5][6][7][8][9][10][11]. The KMN network then in turn mediates the interaction with spindle microtubules to drive chromosome segregation during cell division [12].Previous work identified the histone H3 variant CenH3 (also called CENP-A [13,14]) as a core constituent for defining the site of a functional kinetochore in most eukaryotes. This is because CenH3 forms specialized nucleosomes preferentially found at centromeres that are the target sites for kinetochore assembly [15][16][17][18]. To do so, CenH3 physically in...

Chromosome Segregation: Evolving a Plastic Chromosome–Microtubule Interface

“…Surprisingly, previous work found that a subset of insect species has lost CENP-A, even though other canonical centromere proteins are retained [7]. Now, the work from Cortes-Silva et al [6] provides insights into the molecular composition and organization of Lepidopteran kinetochores, in which both the loss of CENP-A and a gain of holocentromeres have occurred.…”

“…Finally, the plasticity of the kinetochore is also highlighted by changes in the conservation of kinetochore proteins through both substantial changes to their protein sequences and even the loss of selected kinetochore proteins across evolution [4,5]. Now, new work by Cortes-Silva et al [6] in this issue of Current Biology reveals molecular rewiring of kinetochores in holocentric insect species in the order Lepidoptera (which includes butterflies and moths), highlighting changes in kinetochore organization whereby core kinetochore proteins are lost and centromere structure is altered.…”

“…(Lokiarchaeum being the first discovered) that boast possession of several genes that encode what are known as 'eukaryotic signature proteins'. These are genes previously thought not to have homologs in either Bacteria or Archaea [6,7]. Evolutionary microbiologists eagerly await solid demonstrations that the archaeal genes produce proteins that function as they do in eukaryotes [8].…”