Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells

Abstract

The three-dimensional organization of nuclear compartments within living cells determines genome function and yet their underlying self-organizing principles are unclear. We visualize in real-time transcriptionally active compartments (TCs) by the transient enrichment of fluorescently-labeled uridine 5'-triphosphate molecules within living cells. These TCs partially colocalize with active RNA-Pol II in the cell nucleus. Fluorescence anisotropy maps of chromatin compaction evidences a more open chromatin structure at the TCs. Using live-cell timelapse imaging, heterogeneity in the dynamic behavior of TCs has been revealed which falls into three distinct classes: subdiffusive, super-diffusive, and normal diffusive behavior. In contrast, the mobility of a candidate gene locus, either in the repressed or activated state, undergoes a differential restricted motion that is coupled to TC movement. Further TC dynamics is directly affected by small molecule chromatin structure modulators and adenosine triphosphate depletion. This heterogeneous behavior in TC dynamics within living cells could provide an interesting paradigm to explore the spatiotemporal dimension to gene transcription control.

FIGURE 1

Fluorescent labeling of TC in living HeLa cell nucleus: TCs (red) are shown to colocalize with RNA-Pol II antibody (green) in cell nucleus where H2B-EGFP is shown in gray. White line marks out the nuclear boundary Scale bar: 2 μm.

FIGURE 2

Analysis of TC and chromatin compaction: (a) Color-coded anisotropy profile of H2B-EGFP with solid lines marking TCs. (b) A typical line scan across a TC showing the TR-UTP intensity profile (solid, right axis) and the underlying H2B-EGFP anisotropy (r) profile (shaded, left axis). (c) Percentage sites which have underlying chromatin relatively more or less compacted then the neighborhood for TCs sites in HeLa cells under various hours of TSA treatment and for randomly chosen points inside the nucleus. (d) Cumulative distribution of fluorescence anisotropy of the nucleus (solid circles). The shaded bars represent anisotropy distribution below TC foci.

FIGURE 3

Nature of TC dynamics: XY trajectories of various TCs from the same nucleus are shown in solid representation, and in the ATP-depleted cell, are shown in shaded representation.

FIGURE 4

(a) Typical XY trajectories of TCs in Aphidicolin-arrested and TSA-treated cells. (b) Mean α-values for TCs in cells treated with TSA (HDAC inhibitor) or with Aphidicolin (cell-stage specific inhibitor). (c) Corresponding mean velocity for TC. Error bars correspond to standard error.

FIGURE 5

Nature of gene loci mobility: (a) Image of HeLa cell expressing mRFP-lacI-NLS (left panel) and EGFP (middle panel). Scale bar: 5 μm. Arrow indicates position of the EGFP gene locus. Right panel shows a cell where gene loci and TCs are labeled in red and green, respectively. Arrow indicates a colocalization of a gene locus and TCs. Scale bar: 2 μm (b) typical trajectories measured for cells expressing EGFP and under repression. (Inset) Trajectories of a gene locus and TC. (c) Plot of mean α-values of gene loci under various conditions. Error bars correspond to standard error.