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 H3.1(C96A) and H3.1(C110A) 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.

Figure 1

(A) H3.1 sequence conservation between Homo sapiens, Mus musculus, Bos taurus and Rattus norvegicus; (B) Sequences of the H3 variants H3.1, H3.2, H3.3 and the mutants constructed and analysed here. The mutant H3.1^C96S equals H3.2 (red: sequence deviations of H3.1 and H3.3 from H3.2; green: H3.1 mutants studied here); and (C) Nucleosome structure [105,106]. Both H3 copies are displayed in green with C96 in red and C110 in blue. C96 and C110 are located in different α-helices.

Figure 2

Acceptor-bleaching Förster Resonance Energy Transfer (FRET) between CENP-T-EGFP and (A) H3.1-mCherry; (B) H3.2-mCherry and (C) H3.3-mCherry. In one spot in the cell containing kinetochores, the acceptor mCherry is bleached and the donor EGFP fluorescence is measured before and after the bleach. From the difference, the FRET efficiency E_FRET is calculated and grouped according to 4% deviation categories (black bars). Parallel to this measurement, at another spot in the same cell also containing kinetochores, the acceptor is not bleached and the donor EGFP fluorescence is measured before and after the bleach. In this control measurement, the variation of the donor fluorescence is determined at a non-bleached cellular location. This donor fluorescence is treated in the same way resulting in E_VAR (grey bars). For every measurement, the E_FRET distribution was statistically compared to the control E_VAR distribution, obtaining the p-value.

Figure 3

(A) Normalized Fluorescence Recovery After Photobleaching (FRAP) recovery curves of H3.1, H3.1^C96A and H3.1^C110A in S-phase (5 h after double thymidine block release) in transfected HeLa cells. All three proteins show the small and slow recovery typical for H3 [107]; (B,C) Acceptor-bleaching FRET between CENP-T-EGFP and (B) H3.1^C96A-mCherry and (C) H3.1^C110A-mCherry. The large p-values indicate the absence of a FRET signal.

Figure 4

Analysis of myc-tagged H3 variant reverse-phase HPLC fractions. (A) Typical RP-HPLC profile of HeLa histones. Peaks that correspond to individual histones are labelled; (B) Slot immunoblots of RP-HPLC separated histone fractions 50–96 (from top left to bottom right) derived from HeLa cells transfected with indicated myc-tagged H3 wild type histones and mutants. Blots were stained with H2B (top) or myc (bottom) antibodies. H2B detection was chosen as reference elution point; (C) Graphical depiction of fraction numbers between strongest H2B and myc signal seen in (B).

Figure 4

Analysis of myc-tagged H3 variant reverse-phase HPLC fractions. (A) Typical RP-HPLC profile of HeLa histones. Peaks that correspond to individual histones are labelled; (B) Slot immunoblots of RP-HPLC separated histone fractions 50–96 (from top left to bottom right) derived from HeLa cells transfected with indicated myc-tagged H3 wild type histones and mutants. Blots were stained with H2B (top) or myc (bottom) antibodies. H2B detection was chosen as reference elution point; (C) Graphical depiction of fraction numbers between strongest H2B and myc signal seen in (B).

Figure 5

Acceptor-bleaching FRET between EGFP-CENP-M and H3.1-mCherry. The low p-value (p < 0.001) indicates a strong FRET signal.

Figure 6

Boustrophedon kinetochore structure [96]. (A) One loop of a potential centromeric chromatin structure at late S phase. CENP-A dominates one stretch of nucleosomes while H3 variants are distributed on the folded-back strand. The CCAN complex is supposed to fold between a CENP-A on the one and H3.1 on the other strand [99]; (B,C) CENP-T/W/S/X may be wrapped around by DNA in cis to CENP-A-containing (C) or H3.1-containing nucleosomes (B).