Beyond the Nucleosome: Nucleosome-Protein Interactions and Higher Order Chromatin Structure.

The regulation of chromatin biology ultimately depends on the manipulation of its smallest subunit, the nucleosome. The proteins that bind and operate on the nucleosome do so, while their substrate is part of a polymer embedded in the dense nuclear environment. Their molecular interactions must in some way be tuned to deal with this complexity. Due to the rapid increase in the number of high-resolution structures of nucleosome-protein complexes and the increasing understanding of the cellular chromatin structure, it is starting to become clearer how chromatin factors operate in this complex environment. In this review, we analyze the current literature on the interplay between nucleosome-protein interactions and higher-order chromatin structure. We examine in what way nucleosomes-protein interactions can affect and can be affected by chromatin organization at the oligonucleosomal level. In addition, we review the characteristics of nucleosome-protein interactions that can cause phase separation of chromatin. Throughout, we hope to illustrate the exciting challenges in characterizing nucleosome-protein interactions beyond the nucleosome.

Nucleosome binding peptide presents laudable biophysical and in vivo effects.

Chromatin state is highly dependent on the nucleosome binding proteins. Herein, we used a multipronged approach employing biophysical and in vivo experiments to characterize the effects of Nucleosome Binding Peptides (NBPeps) on nucleosome and cell activity. We performed a series of structure-based calculations on the nucleosome surface interaction with GMIP1 (a novel NBPep generated in silico), and HMGN2 (nucleosome binding motif of HMGN2), which contains sites that bind DNA and the acid patch, and also LANA and H4pep (nucleosome binding motif of H4 histone tail) that only bind to the acidic patch. Biochemical assays shows that H4pep, but not HMGN2, GMIP1 and LANA, is highly specific for targeting the nucleosome, with important effects on the final nucleosome structure and robust in vivo effects. These findings suggest that NBPeps might have important therapeutic implications and relevance as tools for chromatin investigation.

Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.