Single and double box HMGB proteins differentially destabilize nucleosomes

Abstract

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A–H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3–H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

“…Nevertheless, linker lengths <30 base pairs have been reported to exist in vivo, depending on cell type and genomic position, [60][61][62][63][64] and are thus frequently used for in vitro measurements 28,34,41,[49][50][51] . However, linker lengths ≥ 30 base pairs can additionally arise in vivo, 61,63,65,66 and can therefore also be relevant for in vitro studies 15,25,27,28,35,38,39,41,[49][50][51] . Such longer linker lengths can be engineered using our approach by simply modifying the first step of the strategy laid out in Fig.…”

“…the mechanical stability of both single nucleosomes and nucleosome arrays, 2,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] as well as probe the influence of remodeling and other enzymes on such substrates 8,[12][13][14][15]17,[36][37][38][39][40][41][42][43] .…”

“…Typically, these arrays consist of repeats of the 601-core sequence flanked by linker DNA that is derived from the 135 base pairs of linker regions associated with the complete 601 sequence. In contrast to mono-nucleosomes, an array of nucleosomes facilitates studying chromatin-like structures, 25,41,[48][49][50]55,57 and can be used to probe the influence of closely spaced nucleosomes on DNA-protein interactions 15,36,40,41 .…”

The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum

“…In other eukaryotic organisms, nonhistone architectural chromosomal proteins, HMGB (high-mobility group B family), have been implicated in the control of gene expression through regulation of local nucleosome dynamics at the promoter regions of a variety of genes. They have also been found to regulate chromosome structure through the action of specific topologically associating domain (TAD) boundaries ( 26 – 28 ). For P. falciparum , four putative HMG proteins (PfHMGB1 to -4) have been predicted by sequence homology.…”

Mechanical properties of nucleoprotein complexes determined by nanoindentation spectroscopy

“…conditions where higher-order folding can be expected to be severely affected yielding variable results [45][46][47][48][49][50][51]. Sub-physiological salt solutions are also often used for fluid AFM operation to get stable imaging of nucleosomes [52,53]. The nucleosomal stability also depends on the core particle concentration [50,51].…”