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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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Every 3617 cell contains at least one MMTV array, and these arrays are visible by GFP-GR in a subset of cells. (a) A low magnification projection image constructed from 16 confocal sections, .28 μm apart, showing 3617 cells that were removed from tetracycline for 16 h and then treated with 100 nM dexamethasone for 1.5 h. Note that the GFP-GR is concentrated in the nucleus of each cell. At least two cells exhibit moderately large MMTV arrays, indicated by the white circles (see McNally et al. [2000] and Figs. 2 and 7 here for a demonstration that these bright structures correspond to the MMTV array). Other nuclei do not reveal obvious array structures, although several exhibit bright structures near nucleoli that may correspond to arrays but cannot be scored definitively at this low magnification. (b) Control DNA FISH with a probe specific for the MMTV array on mouse C127 cells, the grandparent of 3617 that lacks the MMTV array. No DNA FISH signal is detected. Contrast has been amplified in this panel to permit visualization of background staining in the nucleus. (c) DNA FISH with a probe specific for the MMTV array on 3617 cells. Each nucleus shows a distinct FISH signal, indicating that every cell contains at least one copy of the MMTV array. (Sometimes nuclei are observed with two such signals; data not shown.) Bar, 10 μm.
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fig1: Every 3617 cell contains at least one MMTV array, and these arrays are visible by GFP-GR in a subset of cells. (a) A low magnification projection image constructed from 16 confocal sections, .28 μm apart, showing 3617 cells that were removed from tetracycline for 16 h and then treated with 100 nM dexamethasone for 1.5 h. Note that the GFP-GR is concentrated in the nucleus of each cell. At least two cells exhibit moderately large MMTV arrays, indicated by the white circles (see McNally et al. [2000] and Figs. 2 and 7 here for a demonstration that these bright structures correspond to the MMTV array). Other nuclei do not reveal obvious array structures, although several exhibit bright structures near nucleoli that may correspond to arrays but cannot be scored definitively at this low magnification. (b) Control DNA FISH with a probe specific for the MMTV array on mouse C127 cells, the grandparent of 3617 that lacks the MMTV array. No DNA FISH signal is detected. Contrast has been amplified in this panel to permit visualization of background staining in the nucleus. (c) DNA FISH with a probe specific for the MMTV array on 3617 cells. Each nucleus shows a distinct FISH signal, indicating that every cell contains at least one copy of the MMTV array. (Sometimes nuclei are observed with two such signals; data not shown.) Bar, 10 μm.

Mentions: By GFP-GR fluorescence, the MMTV array was observed in the nucleoplasm (Fig. 1 a; a low magnification view) as a brightly labeled structure of varying size and shape (variations described in more detail below). Arrays were observed soon after the addition of hormone (within 10 min), and some arrays were still visible in a fraction of cells (∼7%) up to 32 h later. Arrays were often located next to or in the vicinity of a nucleolus. At 3 h after hormone treatment, typically 30–40% of cells examined at high magnification exhibited an 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)

Every 3617 cell contains at least one MMTV array, and these arrays are visible by GFP-GR in a subset of cells. (a) A low magnification projection image constructed from 16 confocal sections, .28 μm apart, showing 3617 cells that were removed from tetracycline for 16 h and then treated with 100 nM dexamethasone for 1.5 h. Note that the GFP-GR is concentrated in the nucleus of each cell. At least two cells exhibit moderately large MMTV arrays, indicated by the white circles (see McNally et al. [2000] and Figs. 2 and 7 here for a demonstration that these bright structures correspond to the MMTV array). Other nuclei do not reveal obvious array structures, although several exhibit bright structures near nucleoli that may correspond to arrays but cannot be scored definitively at this low magnification. (b) Control DNA FISH with a probe specific for the MMTV array on mouse C127 cells, the grandparent of 3617 that lacks the MMTV array. No DNA FISH signal is detected. Contrast has been amplified in this panel to permit visualization of background staining in the nucleus. (c) DNA FISH with a probe specific for the MMTV array on 3617 cells. Each nucleus shows a distinct FISH signal, indicating that every cell contains at least one copy of the MMTV array. (Sometimes nuclei are observed with two such signals; data not shown.) Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196867&req=5

fig1: Every 3617 cell contains at least one MMTV array, and these arrays are visible by GFP-GR in a subset of cells. (a) A low magnification projection image constructed from 16 confocal sections, .28 μm apart, showing 3617 cells that were removed from tetracycline for 16 h and then treated with 100 nM dexamethasone for 1.5 h. Note that the GFP-GR is concentrated in the nucleus of each cell. At least two cells exhibit moderately large MMTV arrays, indicated by the white circles (see McNally et al. [2000] and Figs. 2 and 7 here for a demonstration that these bright structures correspond to the MMTV array). Other nuclei do not reveal obvious array structures, although several exhibit bright structures near nucleoli that may correspond to arrays but cannot be scored definitively at this low magnification. (b) Control DNA FISH with a probe specific for the MMTV array on mouse C127 cells, the grandparent of 3617 that lacks the MMTV array. No DNA FISH signal is detected. Contrast has been amplified in this panel to permit visualization of background staining in the nucleus. (c) DNA FISH with a probe specific for the MMTV array on 3617 cells. Each nucleus shows a distinct FISH signal, indicating that every cell contains at least one copy of the MMTV array. (Sometimes nuclei are observed with two such signals; data not shown.) Bar, 10 μm.
Mentions: By GFP-GR fluorescence, the MMTV array was observed in the nucleoplasm (Fig. 1 a; a low magnification view) as a brightly labeled structure of varying size and shape (variations described in more detail below). Arrays were observed soon after the addition of hormone (within 10 min), and some arrays were still visible in a fraction of cells (∼7%) up to 32 h later. Arrays were often located next to or in the vicinity of a nucleolus. At 3 h after hormone treatment, typically 30–40% of cells examined at high magnification exhibited an 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