The CENP-T/-W complex is a binding partner of the histone chaperone FACT

Lisa Prendergast,Geneviève Almouzni,Hongda Huang,Yiwei Liu,Sebastian Müller,Kevin F Sullivan,Florent Dingli,Dinshaw J Patel,Isabelle Vassias,Damarys Loew

doi:10.1101/gad.275073.115

Abstract

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

Highlights

The centromere is a specialized chromosomal locus that defines the site of kinetochore assembly
Synthesized CENP-T-CLIP and CENP-W-CLIP accumulation at centromeres occurs in S phase; most EdU-negative cells did not show CENP-T-CLIP or CENP-W-CLIP accumulation at centromeres, in agreement with previous reports
We found that CENP-T deposition at centromeres is uncoupled from DNA synthesis (Fig. 1)

Summary

Introduction

The centromere is a specialized chromosomal locus that defines the site of kinetochore assembly. The functional identity of the centromere is thought to be conveyed by the histone variant CenH3CENP-A (for review, see Black and Cleveland 2011; Müller and Almouzni 2014) This foundation enables nucleation of a core group of 17 additional proteins, the constitutive centromere-associated network (CCAN) necessary for kinetochore function (Foltz et al 2006; Izuta et al 2006; Okada et al 2006). CENP-T plays key roles both in recruiting downstream CCAN components and directly binding to Ndc, the conserved microtubule-binding complex of kinetochores (Gascoigne et al 2011; Hori et al 2012; Malvezzi et al 2013). Several functional interactions of the CENP-T/-W complex at centromeres have been characterized, yet the molecular mechanism enabling CENP-T/-W accumulation at centromeres remains unclear. We propose that FACT acts as a histone chaperone that switches its associated partners between H2A–H2B and CENP-T/-W, regulating CENP-T/-W accumulation at centromeres

Methods

Results

Conclusion