Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

Abstract

Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

The distinct functions of CENP-C and CENP-T/W in centromere propagation and function inXenopusegg extracts

“…Our findings of a different recruitment time for CENP-T and CENP-W could be consistent with the differences in dynamics reported for the 2 proteins in FRAP studies in human cells. 18 In the same study, the fluorescence staining of centromere-associated CENP-T increases by 5-fold from early S-phase to G2 but only 2-fold for CENP-W. Thus, CENP-T could be assembled first onto replicated chromatin and later on recruit CENP-W/S/X.…”

“…14,15 Importantly, while CENP-A nucleosomes are stably bound to centromeric chromatin, 16 the binding of CCAN proteins appears to be dynamic. [17][18][19] CENP-C and CENP-T serve as a platform for recruitment of the KMN network through pathways that are not yet well understood. [20][21][22][23][24][25][26][27][28][29] From the CCAN components, CENP-N and CENP-C recognize CENP-A.…”

“…37,38 How CENP-T/W/S/X are targeted to the centromere is currently unclear although it has been reported that their incorporation occurs in late S phase/G2 and requires CENP-A. 18,35,36,39 It is also unclear whether they contribute to CENP-A deposition.…”

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…One example is the "CLIP-tag", which is derived from SNAP and reacts specifically with a variant substrate, O 2 -benzylcytosine (Gautier et al, 2008). Tagging of two different proteins by SNAP and CLIP allows for simultaneous labeling of two different proteins in different colors (Gautier et al, 2008;Prendergast et al, 2011). More recently, variants of SNAP and CLIP named SNAPf and CLIPf have been developed that present faster reaction kinetics (Pellett et al, 2011;Sun et al, 2011).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…Important insights will come with a more complete understanding of the role of the centromere proteins in maintaining CENP-A nucleosomes across the DNA replication fork, as recent observations show that many centromeric proteins show distinct stability during S phase. CENP-T and CENP-W completely turn over during S phase, but increase in abundance in S/G 2 relative to G 1 (Prendergast et al 2011). CENP-S and CENP-X also assemble at centromeres during S/G 2 (Dornblut et al 2014).…”