Probing the effect of histone variant H2A.Z

Nucleosomes make up the core units of chromatin, and their structural dynamics affect gene expression. The nucleosome core is made up of two copies of each 4 histone proteins H2A, H2B, H3, and H4, with roughly 146 base pairs wrapped around the histone octamer. Histone variants can give rise to differing structural properties, altering the stability of the nucleosomes and the extent of DNA unwrapping. The highly conserved histone variant H2A.Z, together with other regulatory proteins, controls transcription and plays an important role in development and cell differentiation. To evaluate how H2A.Z alters the internal dynamics of nucleosomes containing binding sites of the pioneer transcription factors Sox2 and Oct4, we prepared H2A or H2A.Z nucleosomes with modified Widom 601 nucleosome positioning sequences. Using exonuclease digestion, Förster resonance energy transfer (FRET), and nuclear magnetic resonance (NMR) spectroscopy combined with targeted mutagenesis, we hope to probe what H2A.Z and DNA features are responsible for certain effects. Preliminary results indicate that the DNA sequence and location cause significant changes in dynamics between wild-type and H2A.Z nucleosomes. Exonuclease assays suggest greater DNA unwrapping in the presence of H2A.Z, especially on the TA-rich side of the 601 nucleosome, compared to the TA-poor side. This is supported by altered dynamics in the histone H3 N-terminal tail detected by NMR. Surprisingly, we also observe an interior stabilizing effect with the histone variant. By replacing the H2A.Z N-terminal tail, C-terminal tail, or the L1 loop with those of H2A, we aim to characterize which regions contribute specific effects on the structural dynamics, as well as connect how these changes alter the binding of pioneer transcription factors Sox2 and Oct4.