The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.
16The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely 17 found in chromatin within nuclei. To determine how chromatin high-order structure is 18 controlled, we used cryo-ET to study the undigested natural chromatin released from 19 cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the 20 presence of divalent cations, most of the chromatin from both organisms is compacted 21 into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, 22 some of which include face-to-face interactions. In the absence of divalent cations, 23 picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin 24 mostly remains compact with looser nucleosome packing, even after treatment with 25 histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore 26 sensitive to the local environment, but generally nonpermissive of regular motifs, even 27 at the level of oligonucleosomes.28 70 erythrocytes (Scheffer et al., 2011). These and other studies challenge whether 30-nm 71 fibers is the best model of chromatin within somatic cells (Hansen, 2012;; Nishino et al., 72 2012;; van Holde and Zlatanova, 1995).73 5 The different conclusions between in vitro and in vivo chromatin studies raise the 74 question: can the chromatin of cells that do not show evidence of 30-nm fibers in vivo 75 be made to adopt a 30-nm-fiber structure in vitro? To address this question, we used 76 cryo-ET to study the 3-D organization of undigested natural chromatin from 77 picoplankton and yeast. In the presence of even traces of divalent cations, most of the 78 chromatin in both organisms compacts into large masses. Some of the chromatin does 79 form 30-nm fibers, predominantly of the irregular variety. In the absence of divalent 80 cations, picoplankton chromatin decondenses into open zigzags, but yeast chromatin 81 remains either in a mass or folded as irregular 30-nm fibers. To better understand how 82these nucleosomes interact, we classified the chromatin in both 2-D and 3-D, and found 83 that there is no dominant higher-order packing motif, but the orientation of the DNA at 84 the core-particle entry/exit points is not too variable. Therefore, the higher-order 85 structure of natural chromatin is sensitive to environmental conditions, but this 86 sensitivity varies by species. 87 6 RESULTS 88 89Release of natural picoplankton chromatin 90 To study the higher-order structure of natural picoplankton chromatin in vitro, we 91 lysed cells in hypotonic buffer on ice either with or without divalent cations (Fig. 1B). 92This treatment released all cellular contents, including the chromatin, and is expected to 93 produce thinner plunge-frozen samples that generate higher-contrast cryotomograms. 94Compared to intact cells (Fig. 1C), the lysed cells' contents spread over a much larger 95 area and indeed allowed the ice to be thinner, resulting in higher-contrast tomograms 96 (Fig. 1D). The majority of the densities came from remnants of...
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