Native in situ architecture of the human inactive X chromosome revealed by correlative light and electron microscopy
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Knowledge of the nuclear organization of specific chromatin domains at the ultrastructural level remains limited. To address this, we employed correlative light and electron microscopy (CLEM) approaches to investigate the inactive X chromosome (Xi) in female human RPE1 cells. Tandem fusion of macroH2A (mH2A), a histone variant enriched on the Xi, to eGFP and APEX2 enabled direct labeling and identification of the Xi by light microscopy, transmission electron microscopy, and electron tomography using conventional CLEM. The eGFP tag further allowed us to visualize the Xi at single nucleosome resolution under close-to-native conditions using cryo-CLEM. We found that, although heterochromatin domains of the Xi are substantially larger than those of autosomal regions, their chromatin density was not significantly different. Furthermore, our data reveal the organization of the interchromatin compartment (IC) within the Xi, characterized by a network of larger lacunae and smaller channels. Overall, we provide an ultrastructural characterization of a specific heterochromatin domain at unprecedented resolution, advancing our understanding of 3D chromatin organization and nuclear architecture in situ .
SIGNIFICANCE
How chromatin is organized within the nucleus is a fundamental question in cell and molecular biology. Recent cryo-electron tomography studies have revealed chromatin and nucleosome organization in situ but lack molecular identification of the chromatin domains examined. Here, we investigate the ultrastructure of the human inactive X chromosome (Xi), or Barr body, a highly compact heterochromatin domain, in female RPE1 cells. By combining conventional and cryogenic correlative light and electron microscopy (cryo-CLEM), we characterize its three-dimensional architecture under near-native conditions and resolve chromatin to the level of individual nucleosomes. Our findings reveal the ultrastructure of a defined heterochromatin domain in situ and establish a framework for connecting chromatin molecular identity with native three-dimensional architecture.
