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.