Visualizing chromatin dynamics in intact cells

Abstract

Chromatin and associated regulatory proteins regulate gene expression in the natural environment of the intact cell nucleus. Specific combinations of DNA-binding transcription factors and recruited coregulatory proteins alter the conformation of chromatin at promoters and enhancers of target genes to stimulate or repress transcription. The dynamic nature of the regulatory proteins active in these processes allows the cell to modulate gene expression very rapidly, an important feature in many physiological processes. Live cell imaging and photobleaching studies of fluorescently-tagged proteins reveal that many transcription factors and other chromatin-associated proteins rapidly move through the nucleoplasm. Transcription factors also transiently interact with specific regulatory sequences in chromatin, suggesting that gene activation does not require the formation of stable long-lived regulatory complexes on the chromatin. In this review we discuss how dynamic interactions allow transcriptional regulatory proteins find their targets within the nucleus, alter target chromatin structure, and modulate physiological gene expression.

Fig. 1.

FRAP analysis of protein movement in living cells. The diagram shows laser photobleaching of a small subnuclear region, resulting in loss of fluorescence from GFP-labeled transcription factor (GFP-TF) molecules in the region. (Top) If the GFP-TF is immobile, the region will remain less bright than the surrounding nucleoplasm. Bottom) If the GFP-TF exchanges with the surrounding nucleoplasm then the fluorescence in the region will rapidly return over time. (Right panels) Quantification of the fluorescence signals reveals detailed information about protein dynamics. (Bottom, right) The fluorescence intensity will not fully recover to the prebleach level if a fraction of the GFP-TF is immobile.

Fig. 2.

Direct observation of glucocorticoid receptor binding to response elements. Mammalian cells were engineered to contain multiple tandem copies of an MMTV-LTR reporter construct integrated at a single genomic locus, referred to as the MMTV array [56,57]. These cells harbor 200 copies of a 10 kb promoter–reporter structure in a tandem array with a total length of 2.2×10⁶ bp. The cell derivatives shown here express GFP-GR and Brg1, an ATP-dependent chromatin remodeling protein, labeled with red mCherryFP. Following treatment with 100 nM Dex for 0.5 h, the cells were fixed and processed for RNA FISH to detect the reporter gene and visualized by digital deconvolution microscopy. The images from each fluorescence channel are shown individually (left panels), and merged in the overlay image (far right panel). Arrows show the location of the transcriptionally active MMTV array, where GFP-GR and mCherryFP-Brg1 steady-state concentrations are increased due to specific transient interactions with the MMTV chromatin.

Fig. 3.

Transcription factor movement in an array space during photobleaching. (A) An idealized view of the MMTV array space in 3617 cells is schematically shown. (B) After induction, most of the response elements are occupied with activated glucocorticoid receptor (green filled circles). (C) After the bleach pulse, all receptors in the array space are uniformly bleached (white circles). (D) Many different events are in progress during the recovery phase. New unbleached receptors enter the space and bind to unoccupied response elements. However, some rebinding events will occur with bleached molecules. Furthermore, both bleached and unbleached receptors are leaving the array space.