Biotinylation of lysine 16 in histone H4 contributes toward nucleosome condensation

Abstract

Holocarboxylase synthetase (HLCS) is part of a multiprotein gene repression complex and catalyzes the covalent binding of biotin to lysines (K) in histones H3 and H4, thereby creating rare gene repression marks such as K16-biotinylated histone H4 (H4K16bio). We tested the hypothesis that H4K16bio contributes toward nucleosome condensation and gene repression by HLCS. We used recombinant histone H4 in which K16 was mutated to a cysteine (H4K16C) for subsequent chemical biotinylation of the sulfhydryl group to create H4K16Cbio. Nucleosomes were assembled by using H4K16Cbio and the 'Widom 601' nucleosomal DNA position sequence; biotin-free histone H4 and H4K16C were used as controls. Nucleosomal compaction was analyzed using atomic force microscopy (AFM). The length of DNA per nucleosome was ∼30% greater in H4K16Cbio-containing histone octamers (61.14±10.92nm) compared with native H4 (46.89±12.6nm) and H4K16C (47.26±10.32nm), suggesting biotin-dependent chromatin condensation (P<0.001). Likewise, the number of DNA turns around histone core octamers was ∼17.2% greater in in H4K16Cbio-containing octamers (1.78±0.16) compared with native H4 (1.52±0.21) and H4K16C (1.52±0.17), judged by the rotation angle (P<0.001; N=150). We conclude that biotinylation of K16 in histone H4 contributes toward chromatin condensation.

Figure 1. DNA supercoiling in nucleosomal core particles

Adopted and modified from [30].

Figure 2. Chemical biotinylation and integrity of histone H4

(A) Recombinant native histone H4, mutant H4 (H4K16C), and chemically biotinylated H4 (H4K16Cbio) were probed with anti-biotin. (B) Integrity and equal loading was confirmed using anti-H4.

Figure 3. Purified core histone proteins and nucleosomal DNA

(A) Histone octamers were reconstituted using H4, H4K16C, or H4K16Cbio and stoichiometry was demonstrated using SDS-PAGE and staining with coomassie blue. (B) The size, purity, and integrity of the Widom 601 nucleosomal position sequence was assayed after PCR (a), after extraction with phenol-chloroform precipitation with ethanol (b), and after the final agarose gel purification step (c).

Figure 4. Nucleosomal reconstitution

Successful reconstitution of nucleosomes was confirmed using electrophoretic mobility shift assay and staining with ethidium bromide.

Figure 5. Length of nucleosomal DNA in biotin-defined nucleosomes

Nucleosomes were reconstituted using H4K16Cbio (A), native H4 histone (B), or H4K16C mutant (C) and the length of DNA bound per nucleosome was quantified using AFM.

Figure 6. AFM scans for reconstituted nucleosome particles

Nucleosomes were reconstituted with histones H4K16Cbio (A), H4 (B), and H4K16C (C). Images were acquired using the NanoScope IIId AFM system. Scan sizes are 500nmx500nm. Representation of enlarged AFM scans for nucleosome core reconstituted with histones H4K16Cbio (D), H4 (E), and H4K16C (F). Scan sizes are 200 nm x 200 nm.