Development of multifunctional synthetic nucleosomes to interrogate chromatin-mediated protein interactions.

Various proteins bind to chromatin to regulate DNA and its associated processes such as replication, transcription, and damage repair. The identification and characterization of these chromatin-associating proteins remain a challenge, as their interactions with chromatin often occur within the context of the local nucleosome or chromatin structure, which makes conventional peptide-based strategies unsuitable. Here, we developed a simple and robust protein labeling chemistry to prepare synthetic multifunctional nucleosomes that carry a photoreactive group, a biorthogonal handle, and a disulfide moiety to examine chromatin-protein interactions in a nucleosomal context. Using the prepared protein- and nucleosome-based photoaffinity probes, we examined a number of protein-protein and protein-nucleosome interactions. In particular, we (i) mapped the binding sites for the HMGN2-nucleosome interaction, (ii) provided the evidence for transition between the active and poised states of DOT1L in recognizing H3K79 within the nucleosome, and (iii) identified OARD1 and LAP2α as nucleosome acidic patch-associating proteins. This study provides powerful and versatile chemical tools for interrogating chromatin-associating proteins.

Semisynthesis of site-specifically succinylated histone reveals that succinylation regulates nucleosome unwrapping rate and DNA accessibility.

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.