The Interactions of Centromeric Nucleosomes Elucidated by Atomic Force Microscopy

Author

Shaun Filliaux, University of Nebraska Medical CenterFollow

ORCID ID

0000-0003-0462-5203

Graduation Date

Spring 5-4-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Yuri Lyubchenko

Second Advisor

Don Ronning

Third Advisor

Gloria Borgstahl

Fourth Advisor

Joe Vetro

Abstract

Nucleosomes are the fundamental unit of compaction for DNA in the genome. These positively charged proteins have two main types of nucleosomes: canonical (H3 containing) and centromere (CENP-A containing). The compacting of DNA allows for DNA to fit into the nucleus of cells, but creates a barrier for DNA accessibility for operations such as replication or transcription. Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. Compared to the bulk chromosome, centromeres are composed of H3 and CENP-A nucleosomes in which H3 histones is replaced by its homolog CENP-A histone. This results in nucleosomes with different structures, decreasing the bp of wrapped DNA from 147 bp for H3 nucleosomes to 121 bp for CENP-A nucleosomes. All these factors can contribute to centromere function.

We first studied H3 and CENP-A nucleosomes on dry AFM samples, using a novel three-way junction (3WJ) as a DNA marker, and determined the affinity of mononucleosomes on the 601 sequence, which yielded the result of H3 nucleosomes having a higher binding affinity to the sequence 99% and 92% for H3 and CENP-A nucleosomes, respectively. A similar trend was seen for the dinucleosomes with a lower percentage binding to the 601 for the CENP-A nucleosomes then than H3 nucleosomes. Next, we applied time-lapse, high-speed AFM (HS-AFM) to characterize the dynamics of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ~100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 ones, in which dissociation of the histone core occurs prior to the nucleosome dissociation. The histone core of CENP-A nucleosomes remains intact even after the dissociation of DNA. All of the nucleosomes dynamics studies, indicated a higher stability in CENP-A nucleosome cores vs H3 nucleosomes, a surprising finding due to the lower DNA wrapping of CENP-A nucleosomes.

Transcription is a crucial biological function, and we studied the interaction of H3 and CENP-A nucleosomes separately with NF-κB, one of the critical transcription factors for regulating the immune response and inflammation. We utilized Atomic Force Microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-κB. We found that this transcription factor unravels H3 nucleosomes removing more than 20 base pairs of DNA, and that NF-κB binds to the nucleosome core. Similar results were obtained for the truncated variant of NF-κB comprised only of the Rel Homology domain and missing the transcription activation domain (TAD). This finding suggests that the RelA TAD is not critical in unraveling H3 nucleosomes by NF-κB. By contrast, NF-κB did not bind to or unravel CENP-A nucleosomes. These findings with different affinities for two types of nucleosomes to NF-κB may have implications for understanding the mechanisms of gene expression which are different for the bulk and centromere chromatin.

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 the 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 molecule, 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.

Future studies expanding on this body of work would look at the effect of three or more nucleosomes on a single DNA strand and how the addition of more nucleosomes starts the higher-order compaction, including both H3 nucleosomes and CENP-A nucleosomes to better elucidate the effects both in the centromere and throughout the chromosome. Additionally the effect of NF-κB on dinucleosomes or trinucleosomes could better elucidate what happens in vivo when DNA is completely wrapped by nucleosomes and a transcription factor must access a specific DNA sequence.

Comments

2024 Copyright, the authors

Recommended Citation

Filliaux, Shaun, "The Interactions of Centromeric Nucleosomes Elucidated by Atomic Force Microscopy" (2024). Theses & Dissertations. 820.
https://digitalcommons.unmc.edu/etd/820