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First published online 6 January 2009
doi: 10.1242/jcs.035865

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Combinatorial probabilistic chromatin interactions produce transcriptional heterogeneity

Laboratory of Receptor Biology and Gene Expression, Building 41, B602, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. Transcriptional coregulators concentrate at the MMTV gene array when expressed in stable inducible cell lines. (A,B) Fluorescence microscopy images are shown for (A) the 6643 cell line expressing Ch-Brg1 or (B) the 6790 cell line expressing Ch-GRIP. The images from each fluorescence channel are shown individually (left panels), and merged in the overlay images (far right panels).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Temporal progression of MMTV promoter activity in the averaged cell populations. The parental 3617 cell line, the Ch-Brg1-expressing 6643 cell line, and the Ch-GRIP expressing 6790 cell line were treated with 100 nM Dex for the indicated lengths of time, and processed for RNA FISH. (A-C) Quantitative microscopy techniques measured the levels of (A) RNA FISH signal, (B) GFP-GR array loading ratio, (C) and Ch-coregulator array loading ratio for each cell. The plotted values represent the means of measurements from greater than 300 cells for each condition. The error bars denote the s.e.m.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Cell-to-cell variability during MMTV promoter progression. The single-cell microscopy measurements, which were averaged in Fig. 1, were sorted into histogram bins according to levels of the measured parameters: (A,C,F) RNA FISH signal, (B,D,G) GFP-GR array loading ratio or (E,H) Ch-coregulator array loading ratio in each cell. The stair-step line plots represent the fraction of the cell populations in each histogram bin. Bin size was empirically set for each parameter to highlight the shapes of the distributions. Bin size is 1 for RNA FISH Signal, 0.2 for GFP-array loading ratio, and 0.1 for Ch-coregulator array loading ratio. The plots are grouped by cell line: (A,B) parental 3617 cell line, (C,D,E) the Ch-Brg1-expressing 6643 cell line, and (F,G,H) the Ch-GRIP-expressing 6790 cell line.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Mean noise strength for the cell populations. For each biological condition, a noise-strength value was calculated for individual cells in each replicate culture that was assayed. These noise-strength values from replicate cultures were averaged to generate the plotted mean noise-strength values. Error bars in the plots denote s.e.m. Mean noise-strength values are displayed for the following parameters: (A) FISH signal, (B) GR array loading ratio, and (C) Ch-coregulator array loading ratio.

View larger version (65K): [in this window] [in a new window]   Fig. 5. Pairwise statistical modeling of GR-chromatin association, coregulator-chromatin association and transcriptional output in single cells during MMTV promoter progression. (A) The GFP-GR array loading ratio and RNA FISH signal or (C) Ch-Brg1 array loading ratio and RNA FISH signal were plotted for cell line 6643 populations. For cell line 6790, (B) GFP-GR array loading ratio and RNA FISH signal or (D) Ch-GRIP array loading ratio and RNA FISH signal were also plotted. Each `+' symbol represents the measurements from a single cell, treated with Dex for 0.5 hours (cyan or green) or 4 hours (purple or red).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Relationships between GR-chromatin association and coregulator-chromatin association in single cells during MMTV promoter progression. (A) The GFP-GR array loading ratio and Ch-Brg1 array loading ratio were plotted for cell line 6643 populations. Each `+' symbol represents the measurements from a single cell from the 6643 cell line, treated with Dex for 0.5 hours (cyan) or 4 hours (purple). (B) The GFP-GR array loading ratio and Ch-GRIP array loading ratio were plotted for cell line 6790 populations. Each `+' symbol represents the measurements from a single cell from the 6790 cell line, treated with Dex for 0.5 hours (green) or 4 hours (red).

View larger version (99K): [in this window] [in a new window]   Fig. 7. Distribution of combinatorial GR-array association and cofactor-array association in the cell populations. Local-likelihood statistical methods calculate the probability that an individual cell will concentrate any given level of GFP-GR and coregulator protein (A-B, Ch-Brg1 or D-F, Ch-GRIP) on the MMTV array after 0.5 or 4 hours of Dex treatment (See color bar, red color denotes increased probability). (C,F) Subtracting the probability values at 0.5 hours from the 4 hour probability values shows how the cell distributions change during promoter progression (see color bar).

View larger version (114K): [in this window] [in a new window]   Fig. 8. Multivariate statistical modeling of GR-coregulator-chromatin association and transcriptional output in single cells during MMTV promoter progression. Multivariate local regression was used to calculate the average FISH signal in individual cells that exhibited different combinations of GFP-GR array loading ratios and (A,B) Ch-Brg1 array loading ratios or (D,E) Ch-GRIP array loading ratios. The color indicates the average level of the measured RNA FISH signal in cells with each combination of factor array loading ratios. These values were calculated from cells treated with Dex for (A,D) 0.5 hours or (B,E) 4 hours. The 0.5 hour average FISH values were subtracted from the 4 hour average FISH values to highlight how the relationships between factor array loading ratios and transcriptional output change during promoter progression.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Multiple probabilistic interactions regulate transcription. (A) Many different proteins rapidly exchange over the time scale of seconds with any given target promoter. These proteins include sequence-specific DNA-binding transcription factors such as GR, and diverse coregulator complexes that contain p160 proteins, Swi/Snf chromatin-remodeling proteins, and unknown factors with time-modified activity. (B) The local chromatin environment at the promoter undergoes probabilistic transitions, effecting the balance of rapid factor exchange, and resulting in heterogeneous steady-state binding of each factor in the individual cells. At promoters with complex time-dependent regulation, the probability of the combinatorial interactions changes over minute to hour time scales. (C) The average behavior of entire the cell population over time appears to be highly deterministic at the level of protein-chromatin interaction and transcriptional output.

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