Belmont Lab
 Unversity of Illinois at Urbana-Champaign
Alumni Labs
 
Long-range interphase chromosome movements

Initial work with our lac operator tagged engineered chromosome regions focused on large-scale chromatin structure and changes in this structure ssociated with cell cycle progression and transcriptional activation. However, despite the focus on large-scale chromatin structure we soon documented reproducible changes in nuclear position of these engineered chromosome regions. In two different cell lines, both with a lac operator tagged region located near the nuclear periphery, a careful cell cycle analysis on fixed samples revealed a characteristic change in nuclear positioning to the nuclear interior occurring at a specific time during S phase (S phase chromosome movements). Although this change in intranuclear positioning was quite obvious in statistical analysis of fixed cell populations, initial attempts at live cell imaging failed to show reproducible changes in chromosome position. Long-range movements were observed in small fraction of cells only.

A similar change in intranuclear position from the nuclear periphery to the nuclear interior was observed with both cell lines after tethering of the acidic activation domain of VP16 to the lac operator arrays (changes in gene positioning induced by transcriptional activators ). This change in positoning could be observed even for transcriptionally inactive mutants of VP16. Again initial attempts to visualize this movement in live cells was only partially successful, with movement observed in only a small fraction of cells. A more careful analysis subsequently revealed that the failure to observed consistent long-range movements in live cell imaging was due to an extreme sensitivity of these movements to light exposures. Using exposure conditions which still allowed cell cycle progression essentially killed long-range movements, as demonstrated by control experiments in which statistical analysis of changes in nuclear position after VP16 tethering was done as a function of light exposure. Further experiments using this statistical analysis of nuclear positioning as an assay for phototoxicity identified imaging conditions which allowed extended live cell imaging without perturbation of long-range chromosome movements.

Using these imaging conditions we showed that long-range interphase chromosome movements from the nuclear periphery to the nuclear interior occurred during short bursts of rapid, long-range movement. During these periods, movements approaching one micron per minute could be observed. Interestingly, these movements appeared to be unidirectional along curvilinear paths and dependent, directly or indirectly, on actin and nuclear myosin 1c.

More recently we have described association of the Hsp70 locus with nuclear speckles after heat shock induction of the Hsp70 genes. Initial imaging experiments suggested that in a subset of cells, this association occurred through long-range chromosome movements.

New microscopy technology should provide reduced phototoxicity allowing us to examine these phenomenon more productively in the near future.

 

Publications:

Hu Y, Kireev I, Plutz M, Ashourian N, Belmont AS. Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template. J Cell Biol. 2009 Apr 6; 185 (1) :87-100. PubMed PMID:19349581; PubMed Central PMCID: PMC2700507.

Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS. Long-range directional movement of an interphase chromosome site. Curr Biol. 2006 Apr 18; 16 (8) :825-31. PubMed PMID:16631592.

Tumbar T, Belmont AS. Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator. Nat Cell Biol. 2001 Feb; 3 (2) :134-9. PubMed PMID:11175745.

Li G, Sudlow G, Belmont AS. Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: precise choreography of condensation/decondensation and nuclear positioning. J Cell Biol. 1998 Mar 9; 140 (5) :975-89. PubMed PMID:9490713; PubMed Central PMCID: PMC2132695.

 

 

 

 

 

 

 

 

 

 

 

 

       
 
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