Investigation of parameters of chromatin higher order structure and the making of multiply-acetylated histone H3 via nonsense suppression
Chromatin, is the primary compactor of, as well as a regulator of all functions related to the eukaryotic genome. Chromatin generally is observed to exist in three states in vitro: the 30nm fiber, the 10 nm extended fiber, and the self-associated array particle (oligomers). Historically, the tightly packed 30nm fibers were the most important system in vivo with oligomers having less physiological relevance but in fact strong evidence was never found for the 30nm fiber’s existence in vivo. Technology now exists which is helping to prove that the 30nm fiber is niche behavior for chromatin and in fact in vitro oligomers may help to probe chromatin’s role in regulating genomic functions. Utilizing dynamic light scattering (DLS), differential magnesium induced sedimentation assays, and inductively coupled plasma mass spectrometry, we probed oligomer’s growth kinetics, size, magnesium binding stoichiometry, and threshold magnesium concentration requirement for oligomerization ([Mg2+]50) and looked for changes in their behavior in response to several factors: pH, nucleosomal concentration, magnesium concentration, and array length.First, we found that DLS was a valuable technique for rapidly and accurately detecting size of arrays, intra-array compacted arrays, and array oligomers as well as the growth kinetics of actively growing oligomers. Second, we found that out of all the factors we tested that: 1. Only pH had a strong correlation with [Mg2+]50, 2. Only array concentration had a strong correlation with the growth kinetics of actively oligomerizing particles. 4. Only magnesium concentration had a correlation with magnesium binding stoichiometry and that array may bind an excessive amount of magnesium relative to their charge. Finally, using DLS we have captured preliminary evidence of an intermediately sized array oligomer which is in good agreement with the size of topologically associated domains in vivo. A common post-translational modification (PTM) of proteins is lysine acetylation. This is an especially ubiquitous PTM in the histones of chromatin, and is important for helping to regulate both structural and mechanistic aspects of chromatin. A number of strategies exist for generating acetylated nucleosomes for the in-vitro study of chromatin though they all have various advantages and disadvantages. An especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation in nature can often occur at multiple sites simultaneously, we optimized procedures for recombinantly expressing and purifying histone H3 proteins that incorporates up to four sites of lysine acetylation on the histone tail. Histone octamers containing four sites of lysine acetylation were assembled into mono-nucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex. In the future, it is our hope to combine the use of multiply acetylated histones to probe for what affects their inclusion might have on magnesium binding stoichiometry, size, kinetic growth, and magnesium threshold requirements for self-associated nucleosomal array particles. Any findings might help to differentiate between roles for how these lysine neutralizing PTM’s behave as interactors with distal and proximal nucleosomes vs how their loss may change the electrostatics in the system.