Cryo-ET reveals the macromolecular reorganization of S. pombe mitotic chromosomes in vivo

Abstract

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozenhydrated Schizosaccharomyces pombe cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearestneighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated. chromatin | condensation | cryo-ET | fission yeast C hromatin structure influences key nuclear activities such as transcription, DNA repair, and replication (1). The fundamental unit of chromatin is the nucleosome, which consists of ∼147 bp of DNA wrapped around a histone octamer (2). In mammalian cells, 2-35 nucleosomes pack into irregular "clutches" (3). More than 500 sequential nucleosomes (calculated from the nucleosome repeat length) are thought to interact as topologically associating domains (4). Likewise, in the fission yeast Schizosaccharomyces pombe, some 300-7,000 nucleosomes are thought to associate as compact globular chromatin bodies called domains (5-7). Furthermore, superresolution imaging of S. pombe revealed that the chromatin is not uniformly distributed in vivo (8). These studies suggest that chromatin higher-order structure arises from physical interactions within large groups of nucleosomes. In mitotic cells, chromosomes condense into discrete structures that can be resolved in a light microscope. The factors involved in condensation have been well characterized (9), and a number of models have been considered for the largescale organization of chromatin domains (10). However, the molecular details of chromatin reorganization are still unknown. Knowledge of how chromosomes condense in 3D at the molecular level is needed to explain the nearly global transcriptional repression that happens to the mitotic cells of most eukaryotes (11). Such a model could also explain how a subset of fission yeast genes escape this mitotic repression and get up-regulated (12)(13)(14). Some insights on in vivo chromatin organization were made possible by new methods, including chromatin-conformation capture (Hi-...

Macromolecular and cytological changes in fission yeast G0 nuclei

“…Furthermore, the G0 nucleoplasm appears even denser than the G1 nucleoplasm ( Figure 2, C and D). In both cell types, we did not find any evidence of cryo-ET densities that resemble highly compacted chromatin masses, which are present in the nuclear lysates of both budding and fission yeasts 11,27 .…”

“…We recently used electron cryotomography (cryo-ET) to study S. pombe mitotic chromosome condensation. Our cryotomograms revealed that chromatin compaction as visualized by cryo-ET is less conspicuous than what is seen by fluorescence microscopy 11 . There is no well-defined boundary between condensed chromatin and nucleoplasm because the nucleus is crowded with macromolecular complexes.…”

“…Quantitative analysis of the nuclear macromolecular packing requires knowledge of the identities and positions of the nucleosome-like particles. Thus far, 3-D classification and subtomogram averaging have failed to identify yeast nucleosomes in situ 11 . As a compromise, we attempted 2-D classification 26 on template-matched candidate nucleosome subtomograms, which allows the elimination from consideration of false positives whose size or shape does not match nucleosomes 11 .…”

“…Thus far, 3-D classification and subtomogram averaging have failed to identify yeast nucleosomes in situ 11 . As a compromise, we attempted 2-D classification 26 on template-matched candidate nucleosome subtomograms, which allows the elimination from consideration of false positives whose size or shape does not match nucleosomes 11 . Our 2-D classification did not adequately discriminate nucleosome-like particles because many of the class averages were ambiguous ( Figure S3).…”

Revealing chromatin organization in metaphase chromosomes

“…The question therefore remains whether the observed structures are also prevalent in vivo. Recently, the Gan group applied cryo-ET of macromolecular structures in their native cellular environment to mitotic chromatin of Schizosaccharomyces pombe (Cai et al, 2018). In that case, the authors were not able to detect higher-order organization, but instead found that many nucleosomes were partially unwrapped.…”