Confirmation of Chromonema Fibers

References:

C. Robinett, A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, A. S. Belmont, In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition, J. Cell Biol. 135: 1685-1700 (1996)

Belmont, A.S., A.F. Straight, In vivo visualization of chromosomes using lac operator / repressor binding, (1998) Trends in Cell Biol. 8: 121-124

Background:

Previous work, using a combination light and electron microscopy, led to a chromonema model of interphase and mitotic chromosome organizaton (see Large-scale chromatin structure and chromonema fibers). However, a key component of this work involved visualizaton of large-scale chromatin organization in permeabilized cell nuclei in certain isolation buffers. These isolation buffers were chosen according to the criteria that they preserved the appearance of mitotic chromosomes and interphase nuclei in living cells as assayed by light microscopy. However, this is a low resolution method, and due to problems of overlap from adjacent chromatin, visualization of clear, spatially distinct large-scale chromatin fibers has not been possible in live cells.

 

Results: (Details)

Left Panel: Live cell microscopy shows linear, extended large-scale chromatin fiber which can be traced for over 5 um length as a spatially distinct fiber.

Right Panel: Immunogold staining of these fibers within cells fixed live, without any prior detergent exposure. Because of the nonselective staining of uranyl and lead salts, chromatin structure is difficult to discern within unextracted, traditionally stained cell nuclei. The selective staining provided by the lac operator / repressor system allows visualization of distinct large-scale chromatin fibers from amplified chromosome regions.

Using the lac operator / repressor system combined with gene amplification we created cell lines with large amplified chromosome regions containing lac operator repeats. Stable transformants expressing a GFP-lac repressor-NLS fusion protein allowed direct visualization of these amplified regions in live cells. By exploiting the selective staining of this system, distinct, large-scale chromatin fibers could be visualized unambiguously in live cells and traced as fibers for distances exceeding 5 um in length. Fixation of live cells without any detergent permeabilization, followed by immnogold staining using antibodies against lac repressor allowed visualization by electron microscopy of these large-scale fibers as ~80 nm chromonema fibers, very similar to the chromonema fibers previously described in electron micrographs of permeabilized cell nuclei.

 

Conclusions:

Our results provide clear, unambiguous demonstration of the organization of chromatin into large-scale chromatin fibers within live cells. Although this is seen with amplified chromosome regions, the structures seen are similar to what had been described in earlier work for normal chromosomal regions. We conclude that in these tissue culture, mammalian cells most of the genome is organized into large-scale chromatin fiber segments. Using our approach, we will be able to study the dynamcis of these large-scale chromatin fibers during cell cycle progression.

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