The linker histone H1 is a highly prevalent protein that compacts chromatin and regulates DNA accessibility and transcription. However, the mechanisms behind H1 regulation of transcription factor (TF) binding within nucleosomes are not well understood. Using in vitro fluorescence assays, we positioned fluorophores throughout human H1 and the nucleosome, then monitored the distance changes between H1 and the histone octamer, H1 and nucleosomal DNA, or nucleosomal DNA and the histone octamer to monitor the H1 movement during TF binding. We found that H1 remains bound to the nucleosome dyad, while the C terminal domain (CTD) releases the linker DNA during nucleosome partial unwrapping and TF binding. In addition, mutational studies revealed that a small 16 amino acid region at the beginning of the H1 CTD is largely responsible for altering nucleosome wrapping and regulating TF binding within nucleosomes. We then investigated physiologically relevant post-translational modifications (PTMs) in human H1 by preparing fully synthetic H1 using convergent hybrid phase native chemical ligation. Both individual PTMs and combinations of phosphorylation and citrullination of H1 had no detectable influence on nucleosome binding and nucleosome wrapping, and had only a minor impact on H1 regulation of TF occupancy within nucleosomes. This suggests that these H1 PTMs function by other mechanisms. Our results highlight the importance of the H1 CTD, in particular, the first 16 amino acids, in regulating nucleosome linker DNA dynamics and TF binding within the nucleosome.

Simple and efficient total chemical synthesis of large proteins remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy that should be generally applicable for facile preparation of large proteins. Key to the strategy is the use of sequential ligation on the solid phase for the directed assembly of ~100-residue segments from short, synthetically accessible peptide components. These segments can then be assembled via convergent solution phase ligation, exploiting o-aminoaniline as a chemically flexible cryptic thioester with multiple activation modalitiies on resin and in situ. We demonstrate the feasibility of our approach through the total synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight component peptide segments. We further demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.