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In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes. H1 binds to the linker DNA of nucleosome to form the chromatosome, the next structural unit of chromatin. Structural features on individual chromatosomes contribute to chromatin structure, but not fully characterized. In addition to canonical nucleosomes composed of two copies each of histones H2A, H2B, H3, and H4 (H3 nucleosomes), centromeres chromatin contain nucleosomes in which H3 is replaced with its analog CENP-A, changing structural properties of CENP-A nucleosomes. Nothing is known about the interaction of H1 with CENP-A nucleosomes. Here we filled this gap and characterized the interaction of H1 histone with both types of nucleosomes. H1 does bind both types of the nucleosomes forming more compact chromosome particles with elevated affinity to H3 nucleosomes. H1 binding significantly increases the stability of chromatosomes preventing their spontaneous dissociation. In addition to binding to the entry-exit position of the DNA arms identified earlier, H1 is capable of bridging of distant DNA segments. H1 binding leads to the assembly of mononucleosomes in aggregates, stabilized by internucleosome interactions as well as bridging of the DNA arms of chromatosomes. Contribution of these finding to the chromatin structure and functions are discussed. 

Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-A(nuc)) and H3 nucleosomes (H3(nuc)) and is enriched with alpha-satellite (alpha-sat) DNA repeats. These CENP-A(nuc) have a different structure than H3(nuc), decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3(nuc) to 121 bp for CENP-A(nuc). All these factors can contribute to centromere function. We investigated the interaction of H3(nuc) and CENP-A(nuc) with NF-kappaB, a crucial transcription factor in regulating immune response and inflammation. We utilized atomic force microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-kappaB. We found that NF-kappaB unravels H3(nuc), removing more than 20 bp of DNA, and that NF-kappaB binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-kappaB comprised only of the Rel homology domain and missing the transcription activation domain (TAD), suggesting that RelA(TAD) is not critical in unraveling H3(nuc). By contrast, NF-kappaB did not bind to or unravel CENP-A(nuc). These findings with different affinities for two types of nucleosomes to NF-kappaB may have implications for understanding the mechanisms of gene expression in 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 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. 

Abstract Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability.