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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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Arrays increase in size after hormone treatment and then later decrease. (a) Array size by GFP-GR versus time after hormone. Changes in array size were detected by GFP-GR as a function of time. Cells were treated with 100 nM dexamethasone at time 0 and then fixed in paraformaldehyde at the time points shown. At each time point, 100 cells containing arrays were randomly selected and then the arrays were classified into one of three size ranges. (b) Changes in array size detected by DNA FISH before hormone and 1.5 or 8 h after hormone. For each treatment, 100 cells were randomly selected and then the perimeter of the array in each cell was measured. Note that by 8 h after hormone treatment, arrays have recondensed to the prehormone state. (c) Times of array decondensation and recondensation by live cell analysis. Histogram plot showing times at which arrays showed the first significant signs of either decondensation or condensation. These data were obtained from 128 time-lapse movies of individual cells. On average, decondensation occurred before condensation. Of 22 movies that were long enough to capture both decondensation and condensation, 21 showed a single decondensation followed by a single recondensation (see text).
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fig5: Arrays increase in size after hormone treatment and then later decrease. (a) Array size by GFP-GR versus time after hormone. Changes in array size were detected by GFP-GR as a function of time. Cells were treated with 100 nM dexamethasone at time 0 and then fixed in paraformaldehyde at the time points shown. At each time point, 100 cells containing arrays were randomly selected and then the arrays were classified into one of three size ranges. (b) Changes in array size detected by DNA FISH before hormone and 1.5 or 8 h after hormone. For each treatment, 100 cells were randomly selected and then the perimeter of the array in each cell was measured. Note that by 8 h after hormone treatment, arrays have recondensed to the prehormone state. (c) Times of array decondensation and recondensation by live cell analysis. Histogram plot showing times at which arrays showed the first significant signs of either decondensation or condensation. These data were obtained from 128 time-lapse movies of individual cells. On average, decondensation occurred before condensation. Of 22 movies that were long enough to capture both decondensation and condensation, 21 showed a single decondensation followed by a single recondensation (see text).

Mentions: To determine if the observed differences in array size reflected either a static or a dynamic property of the array, we categorized array size as a function of time after hormone treatment. At 0.5, 1, 3, 6, and 9 h after hormone addition, cells were fixed, and at each time point 100 cells with visible GFP-GR arrays were randomly selected. Each array was then scored as small (a dot-like structure ranging 0–1 μm in diameter as in Fig. 4 a), medium (a cusp-like structure ranging 1–2 μm in length as in Fig. 4 b), or large (a linear or dotted structure with length >3 μm as in Fig. 4, c–l). Fig. 5 a shows the results of such an experiment. The number of large arrays increased over the first 3 h and then decreased back to starting levels by 9 h (red curve). Conversely, the number of small arrays decreased over the first 3 h and then increased back to starting levels by 9 h (blue curve). These observations suggest that in response to hormone treatment, some of the arrays visible by GFP-GR undergo a decondensation and then a subsequent recondensation.


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)

Arrays increase in size after hormone treatment and then later decrease. (a) Array size by GFP-GR versus time after hormone. Changes in array size were detected by GFP-GR as a function of time. Cells were treated with 100 nM dexamethasone at time 0 and then fixed in paraformaldehyde at the time points shown. At each time point, 100 cells containing arrays were randomly selected and then the arrays were classified into one of three size ranges. (b) Changes in array size detected by DNA FISH before hormone and 1.5 or 8 h after hormone. For each treatment, 100 cells were randomly selected and then the perimeter of the array in each cell was measured. Note that by 8 h after hormone treatment, arrays have recondensed to the prehormone state. (c) Times of array decondensation and recondensation by live cell analysis. Histogram plot showing times at which arrays showed the first significant signs of either decondensation or condensation. These data were obtained from 128 time-lapse movies of individual cells. On average, decondensation occurred before condensation. Of 22 movies that were long enough to capture both decondensation and condensation, 21 showed a single decondensation followed by a single recondensation (see text).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196867&req=5

fig5: Arrays increase in size after hormone treatment and then later decrease. (a) Array size by GFP-GR versus time after hormone. Changes in array size were detected by GFP-GR as a function of time. Cells were treated with 100 nM dexamethasone at time 0 and then fixed in paraformaldehyde at the time points shown. At each time point, 100 cells containing arrays were randomly selected and then the arrays were classified into one of three size ranges. (b) Changes in array size detected by DNA FISH before hormone and 1.5 or 8 h after hormone. For each treatment, 100 cells were randomly selected and then the perimeter of the array in each cell was measured. Note that by 8 h after hormone treatment, arrays have recondensed to the prehormone state. (c) Times of array decondensation and recondensation by live cell analysis. Histogram plot showing times at which arrays showed the first significant signs of either decondensation or condensation. These data were obtained from 128 time-lapse movies of individual cells. On average, decondensation occurred before condensation. Of 22 movies that were long enough to capture both decondensation and condensation, 21 showed a single decondensation followed by a single recondensation (see text).
Mentions: To determine if the observed differences in array size reflected either a static or a dynamic property of the array, we categorized array size as a function of time after hormone treatment. At 0.5, 1, 3, 6, and 9 h after hormone addition, cells were fixed, and at each time point 100 cells with visible GFP-GR arrays were randomly selected. Each array was then scored as small (a dot-like structure ranging 0–1 μm in diameter as in Fig. 4 a), medium (a cusp-like structure ranging 1–2 μm in length as in Fig. 4 b), or large (a linear or dotted structure with length >3 μm as in Fig. 4, c–l). Fig. 5 a shows the results of such an experiment. The number of large arrays increased over the first 3 h and then decreased back to starting levels by 9 h (red curve). Conversely, the number of small arrays decreased over the first 3 h and then increased back to starting levels by 9 h (blue curve). These observations suggest that in response to hormone treatment, some of the arrays visible by GFP-GR undergo a decondensation and then a subsequent recondensation.

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