Chromatin in situ

Horowitz, R.A., Agard, D.A., Sedat, J.W., and Woodcock, C.L. (1994) The three-dimensional architecture of chromatin in situ electron tomography reveals fiber composed of a continuously variable zig-zag nucleosomal ribbon. J. Cell. Biol. 125(1), 1-10. 

This chapter focuses on the practical application of imaging techniques to chromatin in situ and in vitro, with emphasis on protocols in use in the authors laboratory. Imaging techniques for the examination of whole nuclei and larger scale chromatin assemblies are discussed by Belmont and by Osheim and Bayer, this volume. 

Because of the superposition of structures in the typical 50-nm thin section, resolution of individual nucleosomes and linker DNA segments in chromatin in situ cannot usually be achieved with single micrographs. Complete three-dimensional reconstruction of tomographic data sets is described by Belmont (see Chapter 6 of this volume), and has been successfully completed for chicken erythrocyte and echinoderm sperm nuclei with OA-B-stained Lowicryl sections (Horowitz et al, 1994). 

This is clearly not the case in chromatin in situ and further investigations are proceeding to quantify and investigate the nature of the breaks involved. 

C. L. Woodcock, Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length. J. Cell Biol. 125, 11-19 (1994). 

Advances in technology and improvements in imaging techniques have provided many approaches for imaging chromatin under in vitro conditions and in situ in fixed or living cells. Some of these techniques are described below. 

Evidence for a mechanism to exchange regulatory factors between the chromatin template and a nucleoplasmic compartment has been provided from imaging transcriptionally active chromatin loci in situ in live cells. Such studies have provided much information on the dynamics of histones and regulatory factors, as well as the large-scale organization of the loci in the context of the nuclear environment. 

The structure of the condensed chromatin fiber is still under discussion [1,23,54], with two competing models the original solenoid model of Finch and Klug [16], and the straight-linker model [12,14,55]. Assessing the structure in vivo or in situ has proven impossible thus far, due to technical limitations. Chromatin fibers released from nuclei into solution by nuclease treatment have been widely used as models for fiber structure such fibers are extended at low ionic strength and condensed at ionic strengths believed to be close to those found in vivo ( 150 mM Na" " or 0.35 mM Mg " "). The salt-induced fiber compaction has been extensively studied in the past but is still poorly understood in terms not only of the details of the structure but also in terms of the molecular mechanisms of the compaction process. 

Fig. 4. In situ hybridization technique. Part of an erythroid precursor cell infected with the human parvovirus B19 and probed for viral DNA. The BI9 nucleic acid is located within the centra electron lucent area of the nucleus (N) and also at nuclear pores (arrowheads). Ch, chromatin M, mitochondrion. A three-step detection protocol was used (see ref. 5 for details). Sheep antidigoxigenin followed by rabbit antisheep Ig and then goat antirabbit Ig conjugated to 10-nm gold. Bar is 0.5 pm.

Shioda et al. [43,44] visualized by electron microscopy both regions of naked DNA and of DNA covered with particles in the chromosome of Halobacterium salinarium isolated from gently lysed cells. In a control experiment, they did not detect such particles in E. coli. They also reported the existence of nucleosome-like structures in S. acidocaldarius and methanogens (unpublished results cited in ref. [43]). The size of the particles detected in H. salinarium (9.5 nm) is similar to that of eukaryotic nucleosomes (10.3 nm) however, this putative archaebacterial chromatin is not as regular as eukaryotic chromatin, since not all of the DNA is covered with nucleosomes and since the length of the DNA spacer between the particles is not uniform. In contrast to these results, Bohrmann and coworkers [45] did not visualize nucleosome-like structures in isolated chromosome fibers of Thermoplasma acidophilum. These authors also reported that in situ the nucleoid of T. acidophilum appears to be highly dispersed in the cytoplasm. 

Hotz, M. A., Gong, J., Traganos, F., and Darzynkiewicz, Z. (1994) Flow cytometric detection of apoptosis comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry 15(3), 237-244. 

A EXPERIMENTAL FIGURE 10-25 Fluorescent-labeled probes hybridized to interphase chromosomes demonstrate chromatin loops and permit their measurement. In situ hybridization of interphase cells was carried out with several different probes specific for sequences separated by known distances in linear, cloned DNA. Lettered circles represent probes. Measurement of the distances between different hybridized probes, which could be distinguished by their color, showed that some sequences (e.g.. A, B, and C), separated from one another by millions of base pairs, appear located near one another within nuclei. For some sets of sequences, the measured distances in nuclei between one probe (e.g., C) and sequences successively farther away initially appear to increase (e.g., D, E, and F) and then appear to decrease (e.g., G and H). The measured distances between probes are consistent with loops ranging in size from 1 million to 4 million base pairs. [Adapted from H. Yokota et al., 1995, J. Cell Biol. 130 1239.]... 

Withdrawal of NGF in primary cultures of neurons induced c-jun and c-myb and, at a later time, c-fos mRNA (Estus et al., 1994). The expression of c-jun was sustained, whereas that of c-fos was transient, occurred around the time of chromatin condensation and was shown by in situ hybridisation to be restricted to neurons exhibiting apoptotic morphology. In this study, NGF deprivation and protein synthesis inhibition had an additive effect on c-jun expression, but blocked c-fos induction. Neutralizing antibodies to c-Jun, c-Fos, FosB, Fra-1 and Fra-2, but not to Jun B or Jun D, protected the neurons from apoptosis. 

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