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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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Time lapse sequences of arrays. Sequences show either no significant change in length (a; ∼33% of movies), moderate length changes (b; ∼26% of movies), or dramatic length changes (c; ∼40% of movies). Time in minutes after addition of 100 nM dexamethasone is shown in the bottom right corner of each image. Bar, 1 μm.
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fig6: Time lapse sequences of arrays. Sequences show either no significant change in length (a; ∼33% of movies), moderate length changes (b; ∼26% of movies), or dramatic length changes (c; ∼40% of movies). Time in minutes after addition of 100 nM dexamethasone is shown in the bottom right corner of each image. Bar, 1 μm.

Mentions: As noted above, 33% of the time-lapse movies revealed no pronounced changes in array size or structure. These relatively static cells (Fig. 6 a) provide a baseline, demonstrating that the array is not subject to large random fluctuations over time in size or structure. Indeed, in all of the time-lapse movies we observed consistent progressive changes in array structure and never observed large random fluctuations in the length of the array. Of those cells exhibiting a condensation change, ∼40% showed formation of moderately sized arrays, defined as those ≤3 μm in length (Fig. 6 b). In ∼60% of the cells exhibiting a condensation change, more dramatic structural changes were observed (Fig. 6 c), defined as the formation of arrays ranging 3–10 μm in length and the concomitant formation of complex structures like those shown in Fig. 4, d–i.


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)

Time lapse sequences of arrays. Sequences show either no significant change in length (a; ∼33% of movies), moderate length changes (b; ∼26% of movies), or dramatic length changes (c; ∼40% of movies). Time in minutes after addition of 100 nM dexamethasone is shown in the bottom right corner of each image. Bar, 1 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Time lapse sequences of arrays. Sequences show either no significant change in length (a; ∼33% of movies), moderate length changes (b; ∼26% of movies), or dramatic length changes (c; ∼40% of movies). Time in minutes after addition of 100 nM dexamethasone is shown in the bottom right corner of each image. Bar, 1 μm.
Mentions: As noted above, 33% of the time-lapse movies revealed no pronounced changes in array size or structure. These relatively static cells (Fig. 6 a) provide a baseline, demonstrating that the array is not subject to large random fluctuations over time in size or structure. Indeed, in all of the time-lapse movies we observed consistent progressive changes in array structure and never observed large random fluctuations in the length of the array. Of those cells exhibiting a condensation change, ∼40% showed formation of moderately sized arrays, defined as those ≤3 μm in length (Fig. 6 b). In ∼60% of the cells exhibiting a condensation change, more dramatic structural changes were observed (Fig. 6 c), defined as the formation of arrays ranging 3–10 μm in length and the concomitant formation of complex structures like those shown in Fig. 4, d–i.

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