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. 2019 Aug 7;11(29):13783-13789.
doi: 10.1039/c9nr02042j. Epub 2019 Jun 18.

The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography

Zhongwu Zhou  1 , Kunpeng Li  2 , Rui Yan  2 , Guimei Yu  2 , Christopher J Gilpin  3 , Wen Jiang  2 , Joseph M K Irudayaraj  4
Affiliations

The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography

Zhongwu Zhou et al. Nanoscale. .

Abstract

The naked DNA inside the nucleus interacts with proteins and RNAs forming a higher order chromatin structure to spatially and temporally control transcription in eukaryotic cells. The 30 nm chromatin fiber is one of the most important determinants of the regulation of eukaryotic transcription. However, the transition of chromatin from the 30 nm inactive higher order structure to the actively transcribed lower order nucleosomal arrays is unclear, which limits our understanding of eukaryotic transcription. Using a method to extract near-native eukaryotic chromatin, we revealed the chromatin structure at the transitional state from the 30 nm chromatin to multiple nucleosomal arrays by cryogenic electron tomography (cryo-ET). Reproducible electron microscopy images revealed that the transitional structure is a branching structure that the 30 nm chromatin hierarchically branches into lower order nucleosomal arrays, indicating chromatin compaction at different levels to control its accessibility during the interphase. We further observed that some of the chromatin fibers on the branching structure have a helix ribbon structure, while the others randomly twist together. Our finding of the chromatin helix ribbon structure on the extracted native chromatin revealed by cryo-ET indicates a complex higher order chromatin organization beyond the beads-on-a-string structure. The hierarchical branching and helix ribbon structure may provide mechanistic insights into how chromatin organization plays a central role in transcriptional regulation and other DNA-related biological processes during diseases such as cancer.

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Conflict of interest statement

Conflicts of interest

There are no conflicts to declare.

Figures

Figure 1
Figure 1
(A) The absorption spectra of extracted chromatin. Chromatin with a concentration (220 ng/μl) was measured twice using Nanodrop. (B) Tangled chromatin exhibits globule-like structure. When directly mounting the extracted chromatin on the continuous carbon coated EM girds without buffer alignment, the positively stained chromatin exhibited a tangled globular-like structure. Bar, 5 μm. (C) Chromatin fibers fold into hierarchically different structures. After mounting the chromatin fiber on the continuous carbon coated EM girds and further using the buffer to align chromatin fibers along a specific direction by placing a filter paper at the edge of the grid to absorb the buffer, the weak force provided by the buffer flow extends the chromatin. The extended chromatin fibers with length as long as 100 μm hierarchically folds into different structures. (D) The beads-on-a-string structure of 10 nm chromatin fibers. Nucleosome-like particles were connected by linker DNA on a replication fork-like structure. The image contrast was reversed in order to present the “nucleosome-like” particles. White dots are gold nanoparticles used as fiducial marker for electron tomography. Bar, 50 nm.
Figure 2
Figure 2
CryoET of transition structure from the 30 nm to the 10 nm chromatin fiber. (A) Cryo-ET image of MCF7 Interphase chromatin with EM stage tilted at 0 degree. Black arrow indicates the root of branching structure while the white arrow indicates one of the branches of the branching structure. The white arrowhead indicates 10 nm gold nanoparticle used as fiducial marker in Cryo-ET. (B) Tomographic slice of the chromatin transition structure. The CryoET data from −56 to +56 degree was reconstructed by IMOD. (C) Image segmentation of the chromatin transition structure using Chimera.
Figure 3
Figure 3
CryoET of transition structure from the 30 nm to 10 nm chromatin fiber and chromatin helix structure. (A) Cryo-ET image of MCF7 Interphase chromatin, tilted at 0 degree. Black arrow indicates the root of the branching structure, and the white arrow indicates one of the branches of the branching structure. The white arrowhead indicates 10 nm gold nanoparticle used as fiducial marker in Cryo-ET. (B) Tomographic slice of the chromatin transition structure. (C) Image segmentation of the chromatin transition structure using Chimera. (D) Observed chromatin helical ribbon structures. Continuous image segmentation of tomography slices using Chimera.
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