Packaging of nucleosomes into heterochromatin is an intricate process as many factors are involved in silencing specific parts of DNA from the transcriptional machinery. Utilizing correlative light and electron microscopy (CLEM) can shed light on this subject as to generate complimentary structural data on how DNA-binding and histone-binding proteins control gene expression. In CLEM, light microscopy can provide wide field images for researchers to identify and localize the region of interest, and then use electron microscopy to zoom in the specific ultracellular structure for higher resolution imaging to further assess the structure of chromatin.
In this project, proteins of interests involve in maintaining heterochromatin domains are tagged with enhanced green fluorescent protein (eGFP) and ascorbate peroxidase 2 (APEX2) for correlating the fluorescent signals from eGFP and the contrast in electron microscopy catalyzed by APEX2. These proteins include linker histone H1.0 and heterochromatin protein 1α (HP1α). H1299 mammalian cells were first transfected with eGFP-APEX2-H1.0 and eGFP-APEX2-HP1α for collecting data in CLEM. The fluorescent images and electron micrographs were correlated, and selected areas of interests of the resin-embedded samples were selected for electron tomographic analysis. Inspection of electron tomograms reveal globular and fibrous chromatin domains enriched in H1.0 and HP1α.