Figure 3.

Heterotypic nucleosomes that contain both Cse4 and H3 can be reconstituted in vitro. (A) Cse4 forms a complex with H3 and H4. Full length Cse4, His₆·H3 and H4 were refolded to a tetramer complex. The sample was purified by Ni-NTA and analyzed by 4–12% Bis–Tris SDS-PAGE gel and Coomassie staining. Lane 1, refolded Cse4–H3·His₆–H4 input (IN); lane 2, unbound Ni-NTA fraction (UB); lane 3, 250 mM imidazole elution (E). Lanes 4, 5 and 6 are samples from refolded untagged Cse4–H3–H4 IN, UB and E, respectively. (B) Refolded H3–H4, Cse4ΔN–H4 or heterotypic tetramers (purified by gel filtration as shown in Supplementary Figure S3A) and H2A–H2B dimers were reconstituted into nucleosomes on 147 bp ‘601’ DNA using salt dilution, and analyzed by 6% native PAGE and ethidium bromide staining. Lane 1, 147 bp ‘601’ DNA; lane 2, Cse4ΔN–His₆·H3–H4₂ tetramers and H2A–H2B; lane 3, His₆·H3–H4 tetramer and H2A–H2B; lane 4, canonical (H3–H4)₂ tetramer and H2A–H2B dimer; lane 5: (Cse4ΔN–H4)₂ tetramer and H2A–H2B dimer. The tetramer used in lane 2 is likely a mixture of His₆·H3–H4, Cse4ΔN–H4 and heterotypic Cse4ΔN–His₆·H3–H4₂ tetramer (Supplementary Figure S3A). (C) Heterotypic nucleosomes contain Cse4ΔN, His₆·H3, H4, H2A and H2B. The nucleosome bands from (B) were excised from the gel, eluted and analyzed by 4–12% Bis–Tris SDS-PAGE and Coomassie staining. Lanes 1 and 2: samples from the nucleosome bands of lanes 2 and 5 of the gel in (B), respectively. (D) Cse4ΔN and His₆·H3 are incorporated into the same nucleosome. Nucleosome samples from (B) were purified from free histones by sucrose-gradient, and the nucleosome fractions were purified by Ni-NTA and analyzed on Bis–Tris 4–12% SDS-PAGE. IN, Ni-NTA input nucleosomes; UB, unbound; W, wash; E, elution fraction. Lanes 1–4: Nuc His₆·H3–Cse4ΔN; nucleosomes assembled from Cse4ΔN–His₆·H3–H4₂ tetramers and H2A–H2B dimers. Lanes 5–8: Nuc Cse4ΔN; nucleosome samples reconstituted from (Cse4ΔN–H4)₂ tetramer and H2A–H2B dimer.