Transcription factor NF-κB unravels nucleosomes

Stormberg, Tommy; Filliaux, Shaun; Baughman, Hannah E.R.; Komives, Elizabeth A.; Lyubchenko, Yuri L.

doi:10.1016/j.bbagen.2021.129934

Cited by 14 publications

(28 citation statements)

References 24 publications

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“…The goal of this study was to reveal nanoscale structural characteristics of nucleosomes assembled on the α-satellite sequence. We utilized Atomic Force Microscopy (AFM) with the capability to characterize nucleosomes on the single molecule level with nanometer resolution to address this issue [ 22 , 23 , 24 , 25 , 26 ]. We assembled CENP-A nucleosomes on three different substrates: a substrate containing an α-satellite motif, a substrate containing non-specific DNA and nucleosomes assembled on the Widom 601 positioning motif to determine whether CENP-A has distinguishing structural characteristics between α-satellite and other DNA sequences.…”

Section: Introductionmentioning

confidence: 99%

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

Stormberg

Lyubchenko

2022

IJMS

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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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Section: Introductionmentioning

confidence: 99%

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

Stormberg

Lyubchenko

2022

IJMS

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“…Nucleosomes were assembled using a gradient dilution method optimized from our previous research. ,− Recombinant human histone octamers were purchased from The Histone Source (Fort Collins, CO) for assembly. Before assembly, histones were dialyzed against the initial dialysis buffer (10 mM Tris pH 7.5, 2 M NaCl, 1 mM EDTA, 2 mM DTT) at 4 °C for 1 h. DNA (25 pmol) was then mixed with the histone octamer at a molar ratio of 1:5.…”

Section: Methodsmentioning

confidence: 99%

Beyond Sequence: Internucleosomal Interactions Dominate Array Assembly

et al. 2022

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The organization of the nucleosome array is a critical component of the chromatin assembly into higher order structure as well as its function. Here, we investigated the contributions of the DNA sequence and internucleosomal interactions on the organization of the nucleosomal arrays in compact structures using atomic force microscopy. We assembled nucleosomes on DNA substrates allowing for the formation of tetranucleosomes. We found that nucleosomes are capable of close positioning with no discernible space between them, even in the case of assembled dinucleosomes. This morphology of the array is in contrast with that observed for arrays assembled with repeats of the nucleosome positioning motifs separated by uniform spacers. Simulated assembly of tetranucleosomes by random placement along the substrates revealed that nucleosome array compaction is promoted by the interaction of the nucleosomes. We developed a theoretical model to account for the role of DNA sequence and internucleosomal interactions in the formation of the nucleosome structures. These findings suggest that, in the chromatin assembly, the affinity of the nucleosomes to the DNA sequence and the strengths of the internucleosomal interactions are the two major factors defining the compactness of the chromatin.

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“…Nucleosomes were assembled on the end-labeled DNA substrate using a gradient dialysis method optimized from our previous research [ 33 , 34 ]. Recombinant human histone octamers were purchased from The Histone Source (Fort Collins, CO, USA) for use in assembly.…”

Section: Methodsmentioning

confidence: 99%

“…The junction arm length analysis was performed by measuring from the free end of the 3WJ to the joint. Flank measurements for nucleosomes were carried out as described previously [ 34 ]. Briefly, the lengths were obtained by measuring from the end of the 3WJ to the center of the nucleosome for the labeled arm and measuring from the center of the nucleosome to the free end of DNA for the non-labeled arm.…”

Section: Methodsmentioning

confidence: 99%

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Sun

Stormberg

Filliaux

et al. 2022

IJMS

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Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

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Transcription factor NF-κB unravels nucleosomes

Cited by 14 publications

References 24 publications

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

Beyond Sequence: Internucleosomal Interactions Dominate Array Assembly

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

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