Analysis of chromatin fibers in Hela cells with electron tomography.

Hongli Feng,Xiaomin Li,Ping Zhu,Lei Sun,Jianguo Zhang

doi:10.1007/s41048-015-0009-9

Abstract

The presence and folding pattern of chromatin in eukaryotic cells remain elusive and controversial. In this study, we prepared ultra-thin sections of Hela cells with three different fixation and sectioning methods, i.e., chemical fixation, high pressure freezing with freeze substitution, and cryo-ultramicrotomy with SEM-FIB (focused ion beam), and analyzed in vivo architecture of chromatin fibers in Hela nuclei with electron tomography technology. The results suggest that the chromatin fibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm.

Highlights

The folding of chromatin in eukaryotic cells is closely related to the genetic transcription, replication and repair (Horn and Peterson 2002; Luger et al 1997)
The nucleosome had been structurally characterized by X-ray crystallography at 1.9 Å (Davey et al 2002), how polynucleosomes are folded into 30-nm chromatin fibers, which are typically regarded as the secondary structure of DNA, is inconclusive
We have examined in situ architecture of chromatin fibers in eukaryotic Hela cells with ET analysis based on three different sample preparing technologies, i.e., conventional chemical fixation, high-pressure freezing and freeze substitution, and fast frozen and cryo-focused ion beam (FIB) sectioning

Summary

Introduction

The folding of chromatin in eukaryotic cells is closely related to the genetic transcription, replication and repair (Horn and Peterson 2002; Luger et al 1997). The linear ‘‘beads-on-string’’ arrangement of nucleosomes, which is formed by histone octamers (H2A:H2B: H3:H4 = 2:2:2:2) (Luger et al 1997) wrapped by DNA, is regarded as the first level arrangement of chromatin (Huynh et al 2005). The nucleosome had been structurally characterized by X-ray crystallography at 1.9 Å (Davey et al 2002), how polynucleosomes are folded into 30-nm chromatin fibers, which are typically regarded as the secondary structure of DNA, is inconclusive. The existence of 30-nm chromatin fibers in the nuclei of eukaryotic cells is still remained to be elucidated in vivo (Eltsov et al 2008)

Methods

Results

Conclusion