CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.
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Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy
Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length
- Award ID(s):
- 2303862
- NSF-PAR ID:
- 10523080
- Publisher / Repository:
- Frontiers in Molecular Biosciences
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 9
- ISSN:
- 2296-889X
- Page Range / eLocation ID:
- 1106588
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The interaction of positively charged N‐terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post‐translational modifications and recognition by chromatin‐binding proteins. Here, we report residue‐specific15N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2‐μs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2022.1106588
