The CENP-T C-Terminus Is Exclusively Proximal to H3.1 and not to H3.2 or H3.3

Abstract

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere–kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1C96A and H3.1C110A nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2.

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

“…This is consistent with recent reports that predict that CENP-T/-W and CENP-S/-X are part of separate protein cohorts within the kinetochore (Samejima et al 2015). In agreement with previous reports regarding CENP-T/-W interactions, we did not find CenH3 CENP-A in our CENP-W-GFP pull-downs (Hori et al 2008;Abendroth et al 2015), confirming the specificity of our approach. The presence of both FACT chaperone complex subunits (SSRP1 and Spt16) in both the low-salt and high-salt sample fractions of our MS analysis caught our attention, especially given that FACT has previously been reported as a component of centromeric chromatin pull-downs where CENP-T (also annotated as ICEN22/ FLJ13111) was also found (Foltz et al 2006;Izuta et al 2006).…”

Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function

“…This substitution could trigger changes in molecular properties of H3.1, which makes it distinguishable from H3.2, as evident by a specific elution profile using HPLC. A possible explanation may be that this residue is a site for post-translational modification, and therefore, could alter the structure and/or dynamics ( 60–62 ). Another study has shown that this substitution can significantly increase the stability of the tetramer.…”

Imaging from anatomic to molecular and atomic resolution scales: A review

“…Recently, the technique has demonstrated, to be a valuable tool in our study the centromeric chromatin (Llères et al, 2009). This is the chromatin site where the kinetochore structure is anchored by the presence of CENP-A histone and longer links made possible by the CENP-C terminal tails as we have reported in Abendroth et al (2015). A combination of the STORM and FRET in the study kinetochore structure and centromeric chromatin is expected to be a major breakthrough in understanding of these highly dynamic structures in the cell cycle.…”