In Vivo Tagging of Chromosome Sites and Regions

References:

Belmont, A.S. (2001) "Visualizing chromosome dynamics with GFP," Trends in Cell Biology 11:250-257.

Belmont, A.S., Li, G., Sudlow, G., Robinett, C., Visualization of large-scale chromatin structure and dynamics using the lac operator / lac repressor reporter system, Methods Cell Biol.58: 203-222 (1998)

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

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)

Background:

More than 100 years ago, the dynamic behavior of mitotic chromosomes captured the interest of early microscopists. Using transmitted light microscopy without any special staining, mitotic chromosome segregation can be observed directly in living cells over a time scale of seconds to minutes. The ability to visualize this dynamic process has been crucial to dissecting the molecular mechanisms underlying chromosome segregation.

Conversely, the inability to easily visualize chromosomes in various organisms has led to serious scientific roadblocks. The inability to visualize bacterial chromosomes easily has meant that chromosome segregation in prokaryotic cells has remained a "black box" for many decades. Even in eukaryotic organisms, the inability to easily visualize mitotic chromosomes, for instance in budding and fission yeast, has hindered the full exploitation of the genetic tools available for these model organisms.

More significantly, visualization of decondensed, diploid interphase chromosomes has remained technically challenging in all eukaryotic organisms. Basic questions of interphase chromosome structure as a result have relied largely on static images from fixed material and have been plagued by concerns over specimen preparation artifacts. It is not too great a stretch to conclude that this inability to visualize easily chromosome regions within interphase nuclei has contributed to the relative backwardness of the cell biology of many aspects of nuclear structure and function.

Several years ago, our laboratory set out to develop an in vivo method for tagging specific chromosome regions. The idea was to replace in situ hybridization techniques, dependent on DNA denaturation, with alternative methods using sequence specific protein binding. As our initial system we used the lac operator / lac repressor DNA/protein recognition system from E. Coli.

Results: (Details)

We used a lac operator direct repeat as our DNA tag. Our initial constructs contained 256 copies, totaling ~10.1 kb in length. The operator array was detected initially by staining of fixed samples with purified lac repressor protein or expression in vivo of a lac repressor-NLS (nuclear localization signal) fusion protein followed by immunostaining. The timely introduction of green fluorescent protein (GFP) greatly facilitated in vivo labeling through the development of a GFP-repressor-NLS fusion protein. Alternatively we have introduced purified lac repressor protein, labeled with GFP or covalently attached fluorochromes, into cells by microinjection or bead loading. Deletion of 5-11 C-terminal amino acids of the lac repressor prevents tetramer formation, leading to dimers that bind to single operators. Use of this deletion has improved the stability of lac operator repeats in mammalian and yeast cells.

We applied this methodology initially in mammalian CHO cells to detect single insertions of our 256 copy, ~10.1 kb lac operator array. More recently we have extended this approach to Drosophila melanogaster, in which we have been able to detect witin diploid imaginal disk nuclei operator arrays as small as 700 bp in size. In collaboration with Andrew Murray's laboratory (now at Harvard) this approach was extended to budding yeast, and in collaboration with Richard Losick's laboratory (Harvard) this approach was extended to prokaryotic cells.

Examples of detection of tagged chromosomal sites using the lac operator / repressor system. Detection of single insertions of the 256 copy lac operator array is shown in budding yeast (A), B. subtilis bacteria (B), and mammalian CHO cells (C). In (A) and (C), accumulation of GFP-lac repressor protein within nuclei, due to the NLS of the fusion protein, allows nuclei to be visualized. The small, bright spots in each example (arrows in A, green spots in B, and bright spot, shown in insets, in C) correspond to the diffraction limited lac operator array. In (A) a transmitted light image is superimposed on the fluorescent, green image of the GFP-repressor distribution.

Conclusions:

Our results demonstrate the feasibility of labeling discrete chromosomal sites using DNA-protein recognition. This has been done in both single cell organisms and multi-cellular metazoans, such as Drosophila. This approach opens a number of new avenues for visualization of chromosome dynamics. We also envision the use of this approach in designing direct visual screens for the application of genetic, reverse genetic, and chemical genetic screens to investigate many aspects of chromosome structure and dynamics.