Cryo-Electron Tomography Reveals the Multiplex Anatomy of Condensed Native Chromatin and Its Unfolding by Histone Citrullination

Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast

“…The handle connecting doublets also shows interesting features. Its width is remarkably consistent when compared to the widths of the rest of the kinetochore and its dimensions are not far from what might be expected from a 30 nm chromatin fiber, especially at the salt concentrations present in our buffers 61 . In some cases, the handles also show a ring-like gap, potentially indicating some kind of linkage or hinge point.…”

“…Second, compaction of the chromatin surrounding the centromeric nucleosome could also create a dense environment that obscures the CCAN. Consistent with this, chromatin has been shown to tightly compact at magnesium chloride concentrations similar to those present in the purification buffer 61 . We also were unable to discern the KMN network in the tomograms.…”

Nanoscale analysis of human G1 and metaphase chromatinin situ

“…This asymmetry is also evident in the in situ class averages of HeLa and budding yeast cells (Cai et al ., 2018a; Tan et al ., 2023). Nucleosomes with asymmetric linker DNA structure have been seen in cryo-EM studies of nucleosomes with histones and DNA from cellular lysates (Arimura et al , 2021); reconstituted oligonucleosomes within small condensates (Zhang et al ., 2022); digested native oligonucleosomes (Jentink et al , 2023); decompacted isolated mitotic chromosomes (Beel et al ., 2021); and reconstituted chromatosomes (Bednar et al , 2017; Wang et al , 2021b; Zhou et al , 2021). In contrast, nucleosomes with symmetric linker DNA have been found in vitro in oligonucleosome arrays assembled from the Widom “601” sequence and that have more than two ordered nucleosomes (Dombrowski et al ., 2022; Ekundayo et al , 2017; Garcia-Saez et al ., 2018; Jentink et al ., 2023; Lewis et al , 2021; Schalch et al , 2005; Song et al ., 2014; Zhou et al , 2022).…”

“…Nucleosomes with asymmetric linker DNA structure have been seen in cryo-EM studies of nucleosomes with histones and DNA from cellular lysates (Arimura et al , 2021); reconstituted oligonucleosomes within small condensates (Zhang et al ., 2022); digested native oligonucleosomes (Jentink et al , 2023); decompacted isolated mitotic chromosomes (Beel et al ., 2021); and reconstituted chromatosomes (Bednar et al , 2017; Wang et al , 2021b; Zhou et al , 2021). In contrast, nucleosomes with symmetric linker DNA have been found in vitro in oligonucleosome arrays assembled from the Widom “601” sequence and that have more than two ordered nucleosomes (Dombrowski et al ., 2022; Ekundayo et al , 2017; Garcia-Saez et al ., 2018; Jentink et al ., 2023; Lewis et al , 2021; Schalch et al , 2005; Song et al ., 2014; Zhou et al , 2022). Taking into account the previous cryo-EM studies, our in situ data, and mesoscale simulations (Collepardo-Guevara & Schlick, 2014), we suggest that in situ linker DNA conformational variability may suppress the formation of ordered oligonucleosome structures that have more than two nucleosomes.…”

“…Nucleosome stacking has been observed in multiple contexts, with varying degrees of order. Stacking is found in preparations of mononucleosomes (Bilokapic et al , 2018; Dubochet & Noll, 1978; Eltsov et al ., 2018; Zhou et al , 2018); reconstituted nucleosome arrays (Dombrowski et al ., 2022; Ekundayo et al ., 2017; Garcia-Saez et al ., 2018; Geiss et al , 2014; Jentink et al ., 2023; Lewis et al ., 2021; Schalch et al ., 2005; Song et al ., 2014; Takizawa et al , 2020; Zhou et al ., 2022); isolated natural chromatin ( ex vivo ) from chicken erythrocytes, starfish spermatozoids, picoplankton, and budding yeast (Cai et al ., 2018c; Scheffer et al , 2012); isolated chicken erythrocyte nuclei (Scheffer et al ., 2011); in situ in interphase HeLa, T-lymphoblast, KE37, non-mitotic fly brain, and fly embryo cells (Cai et al ., 2018a; Eltsov et al ., 2018; Fatmaoui et al ., 2022; Hou et al ., 2023). In contrast, nucleosome stacking was not observed in either budding or fission yeast in situ (Cai et al ., 2018b; Tan et al ., 2023).…”