Gene-scale in vitro reconstitution reveals histone acetylation directly controls chromatin architecture

Abstract

Understanding how epigenetic modifications intrinsically shape gene-scale chromatin architecture remains challenging due to difficulties in reconstituting and characterizing sufficiently long arrays with defined modification patterns. Here, we overcome this barrier by reconstituting 20-kilobase (96-nucleosome) chromatin arrays with modification patterns precisely controlled at 12-nucleosome resolution. Single-molecule microscopy reveals the dynamics governed by hydrodynamic interactions, demonstrating that increasing histone H4 acetylation density enhances structural fluctuations and relaxation times. In vitro Hi-C reveals power-law decay of the nucleosome contacts consistent with the Gaussian chain, which is globally reduced by acetylation. We also observe that heterogeneous modification patterns alone are sufficient to create distinct structural domains reminiscent of higher-order chromatin organization. These findings establish how histone modifications modulate chromatin architecture via changes in local stiffness and nucleosome interactions, providing a quantitative framework for genome organization.