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Abstract The formation of condensed heterochromatin is critical for establishing cell-specific transcriptional programs. To reveal structural transitions underlying heterochromatin formation in maturing mouse rod photoreceptors, we apply cryo-electron microscopy (cryo-EM) tomography, AI-assisted denoising, and molecular modeling. We find that chromatin isolated from immature retina cells contains many closely apposed nucleosomes with extremely short or absent nucleosome linkers, which are inconsistent with the typical two-start zigzag chromatin folding. In mature retina cells, the fraction of short-linker nucleosomes is much lower, supporting stronger chromatin compaction. By cryo-EM-assisted nucleosome interaction capture, we observe that chromatin in immature retina is enriched with i ± 1 interactions, while chromatin in mature retina contains predominantly i ± 2 interactions typical of the two-start zigzag. By mesoscale modeling and computational simulation, we clarify that the unusually short linkers typical of immature retina are sufficient to inhibit the two-start zigzag and chromatin compaction by the interference of very short linkers with linker DNA stems. We propose that this short linker composition renders nucleosome arrays more open in immature retina and that, as the linker DNA length increases in mature retina, chromatin becomes globally condensed via tight zigzag folding. This mechanism may be broadly utilized to introduce higher chromatin folding entropy for epigenomic plasticity. 

Centromeres are marked by the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle, the constitutive centromere-associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome conformations in vivo is unknown. Here, we purify endogenous centromeric chromatin associated with the CENP-C complex across the cell cycle and analyze the structures by single-molecule imaging and biochemical assays. CENP-C complex–bound chromatin was refractory to MNase digestion. The CENP-C complex increased in height throughout the cell cycle culminating in mitosis, and the smaller CENP-C complex corresponds to the dimensions of in vitro reconstituted constitutive centromere-associated network. In addition, we found two distinct CENP-A nucleosomal configurations; the taller variant was associated with the CENP-C complex. Finally, CENP-A mutants partially corrected CENP-C overexpression–induced centromeric transcription and mitotic defects. In all, our data support a working model in which CENP-C is critical in regulating centromere homeostasis by supporting a unique higher order structure of centromeric chromatin and altering the accessibility of the centromeric chromatin fiber for transcriptional machinery.