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Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter.

Müller WG, Walker D, Hager GL, McNally JG - J. Cell Biol. (2001)

Bottom Line: Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2).These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin.They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, 41 Library Dr., Bethesda, MD 20892, USA.

ABSTRACT
We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a approximately 0.5-microm spot to form a fiber 1-10 microm long. Arrays later recondensed by 3-8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

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Transcription is required for decondensation. (a) Shown are the percentage of large arrays (>3 μm in length) observed at different DRB concentrations after two different treatment regimens. The data labeled “DRB before Decondensation” come from cells treated simultaneously with 100 nM dexamethasone and different concentrations of DRB. After 2 h, the number of large decondensed arrays was counted. Note that large decondensed arrays decreased significantly in the DRB-treated cells. The cells labeled “DRB after Decondensation” were first treated with 100 nM dexamethasone for 1.5 h to allow arrays to decondense. Then DRB was added at the concentrations indicated, and after a 30–min incubation the percentage of large arrays was determined. Note the significant closing of arrays after the short DRB treatment. Similar results were obtained with α-amanitin. To permit α-amanitin entry, cells were pretreated for 2 h with the drug at the concentrations indicated, and then 100 nM dexamethasone was added for 1.5 h and the number of large arrays was counted. (b) Distribution of array sizes before and after DRB. The effects of DRB on recondensation were not specific to large arrays. Array perimeters were measured from 100 randomly selected cells, each treated with 100 nM hormone for 2 h and then fixed in paraformaldehyde. This distribution was compared with perimeters from cells treated identically except for the addition of 100 μg/ml DRB in the last half hour before fixation. The perimeter distribution after DRB (red, ▪) is shifted to smaller sizes. (c) Comparison of array sizes after DRB to before hormone. A comparable shift in the perimeter distribution occurs when DRB is added with hormone (green curve, ▴). Whether DRB is added with or 1.5 h after hormone, the perimeter distribution (assayed by GFP-GR) resembles that before hormone was added (blue curve, ♦; assayed by DNA FISH). This demonstrates that DRB treatment induces condensation to the prehormone state.
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fig8: Transcription is required for decondensation. (a) Shown are the percentage of large arrays (>3 μm in length) observed at different DRB concentrations after two different treatment regimens. The data labeled “DRB before Decondensation” come from cells treated simultaneously with 100 nM dexamethasone and different concentrations of DRB. After 2 h, the number of large decondensed arrays was counted. Note that large decondensed arrays decreased significantly in the DRB-treated cells. The cells labeled “DRB after Decondensation” were first treated with 100 nM dexamethasone for 1.5 h to allow arrays to decondense. Then DRB was added at the concentrations indicated, and after a 30–min incubation the percentage of large arrays was determined. Note the significant closing of arrays after the short DRB treatment. Similar results were obtained with α-amanitin. To permit α-amanitin entry, cells were pretreated for 2 h with the drug at the concentrations indicated, and then 100 nM dexamethasone was added for 1.5 h and the number of large arrays was counted. (b) Distribution of array sizes before and after DRB. The effects of DRB on recondensation were not specific to large arrays. Array perimeters were measured from 100 randomly selected cells, each treated with 100 nM hormone for 2 h and then fixed in paraformaldehyde. This distribution was compared with perimeters from cells treated identically except for the addition of 100 μg/ml DRB in the last half hour before fixation. The perimeter distribution after DRB (red, ▪) is shifted to smaller sizes. (c) Comparison of array sizes after DRB to before hormone. A comparable shift in the perimeter distribution occurs when DRB is added with hormone (green curve, ▴). Whether DRB is added with or 1.5 h after hormone, the perimeter distribution (assayed by GFP-GR) resembles that before hormone was added (blue curve, ♦; assayed by DNA FISH). This demonstrates that DRB treatment induces condensation to the prehormone state.

Mentions: The correlation between the MMTV array decondensation and transcriptional response could arise because transcription induces decondensation, because decondensation facilitates transcription, or some combination of these two. To test the role of transcription, cells were treated with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a protein kinase inhibitor that blocks the transition from polymerase II initiation to elongation (Chodosh et al., 1989; Marshall and Price, 1992). By performing RNA FISH on DRB-treated cells, we found that 1 μg/ml DRB had no effect on transcript levels at the array, whereas 25 μg/ml reduced these levels, and 100 μg/ml abolished them (data not shown). When cells were treated for 2 h with hormone and 25 or 100 μg/ml DRB, the number of large arrays showed significant decreases that were related to the level of transcript inhibition (Fig. 8 a). These results suggest that transcriptional elongation is required for decondensation of the MMTV array.


Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter.

Müller WG, Walker D, Hager GL, McNally JG - J. Cell Biol. (2001)

Transcription is required for decondensation. (a) Shown are the percentage of large arrays (>3 μm in length) observed at different DRB concentrations after two different treatment regimens. The data labeled “DRB before Decondensation” come from cells treated simultaneously with 100 nM dexamethasone and different concentrations of DRB. After 2 h, the number of large decondensed arrays was counted. Note that large decondensed arrays decreased significantly in the DRB-treated cells. The cells labeled “DRB after Decondensation” were first treated with 100 nM dexamethasone for 1.5 h to allow arrays to decondense. Then DRB was added at the concentrations indicated, and after a 30–min incubation the percentage of large arrays was determined. Note the significant closing of arrays after the short DRB treatment. Similar results were obtained with α-amanitin. To permit α-amanitin entry, cells were pretreated for 2 h with the drug at the concentrations indicated, and then 100 nM dexamethasone was added for 1.5 h and the number of large arrays was counted. (b) Distribution of array sizes before and after DRB. The effects of DRB on recondensation were not specific to large arrays. Array perimeters were measured from 100 randomly selected cells, each treated with 100 nM hormone for 2 h and then fixed in paraformaldehyde. This distribution was compared with perimeters from cells treated identically except for the addition of 100 μg/ml DRB in the last half hour before fixation. The perimeter distribution after DRB (red, ▪) is shifted to smaller sizes. (c) Comparison of array sizes after DRB to before hormone. A comparable shift in the perimeter distribution occurs when DRB is added with hormone (green curve, ▴). Whether DRB is added with or 1.5 h after hormone, the perimeter distribution (assayed by GFP-GR) resembles that before hormone was added (blue curve, ♦; assayed by DNA FISH). This demonstrates that DRB treatment induces condensation to the prehormone state.
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Related In: Results  -  Collection

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fig8: Transcription is required for decondensation. (a) Shown are the percentage of large arrays (>3 μm in length) observed at different DRB concentrations after two different treatment regimens. The data labeled “DRB before Decondensation” come from cells treated simultaneously with 100 nM dexamethasone and different concentrations of DRB. After 2 h, the number of large decondensed arrays was counted. Note that large decondensed arrays decreased significantly in the DRB-treated cells. The cells labeled “DRB after Decondensation” were first treated with 100 nM dexamethasone for 1.5 h to allow arrays to decondense. Then DRB was added at the concentrations indicated, and after a 30–min incubation the percentage of large arrays was determined. Note the significant closing of arrays after the short DRB treatment. Similar results were obtained with α-amanitin. To permit α-amanitin entry, cells were pretreated for 2 h with the drug at the concentrations indicated, and then 100 nM dexamethasone was added for 1.5 h and the number of large arrays was counted. (b) Distribution of array sizes before and after DRB. The effects of DRB on recondensation were not specific to large arrays. Array perimeters were measured from 100 randomly selected cells, each treated with 100 nM hormone for 2 h and then fixed in paraformaldehyde. This distribution was compared with perimeters from cells treated identically except for the addition of 100 μg/ml DRB in the last half hour before fixation. The perimeter distribution after DRB (red, ▪) is shifted to smaller sizes. (c) Comparison of array sizes after DRB to before hormone. A comparable shift in the perimeter distribution occurs when DRB is added with hormone (green curve, ▴). Whether DRB is added with or 1.5 h after hormone, the perimeter distribution (assayed by GFP-GR) resembles that before hormone was added (blue curve, ♦; assayed by DNA FISH). This demonstrates that DRB treatment induces condensation to the prehormone state.
Mentions: The correlation between the MMTV array decondensation and transcriptional response could arise because transcription induces decondensation, because decondensation facilitates transcription, or some combination of these two. To test the role of transcription, cells were treated with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a protein kinase inhibitor that blocks the transition from polymerase II initiation to elongation (Chodosh et al., 1989; Marshall and Price, 1992). By performing RNA FISH on DRB-treated cells, we found that 1 μg/ml DRB had no effect on transcript levels at the array, whereas 25 μg/ml reduced these levels, and 100 μg/ml abolished them (data not shown). When cells were treated for 2 h with hormone and 25 or 100 μg/ml DRB, the number of large arrays showed significant decreases that were related to the level of transcript inhibition (Fig. 8 a). These results suggest that transcriptional elongation is required for decondensation of the MMTV array.

Bottom Line: Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2).These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin.They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, 41 Library Dr., Bethesda, MD 20892, USA.

ABSTRACT
We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a approximately 0.5-microm spot to form a fiber 1-10 microm long. Arrays later recondensed by 3-8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

Show MeSH
Related in: MedlinePlus