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Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

Bottom Line: We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models.Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes.Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

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3-D visualization of an ∼250-nm wide subunit spanning a fraction of the chromatid width supports a hierarchical folding model. (A) Dependence of labeled band width versus insert size reveals folding subunits. In a simple radial loop model (top), as the insert size increases, the labeled region spans an increasing fraction of the chromatid cross section, with the minimal width of a labeled band corresponding roughly to the diameter of a 30-nm chromatin fiber loop. With a successive coiling model, as the insert region increases in size, the labeled region spans an increasing fraction of the chromatid cross section, but the width of this labeled region does not increase until it spans a full chromatid cross section (bottom). The width of the minimal labeled region corresponds to the diameter of the folding subunit, significantly larger than a 30-nm chromatin loop. (B) Reconstructed orthogonal cross sections of EM serial section data built with NewVision. Arrows and arrowhead show the same nanogold-labeled areas as in Fig. 8, I–L. The spot-like labeled region appears as a labeled band extending across the chromatid in the orthogonal view. Thick red, green, and blue lines in images a, b, and c are, respectively, X, Y, and Z axes of a left orthogonal system with origin inside the nanogold-labeled insert. (C) Solid model display for same chromosome region shown in Fig. 8, I–L and Fig. 9 B reveals similar appearance of labeled regions on both chromatids. In the original serial sections, one region appeared as a band (Fig. 8 I, arrowhead) and one as a spot (Fig. 8, J and K, arrows). In the orthogonal views in B and in the solid model, both regions appear as ∼250–300-nm wide segments spanning only a fraction of the chromatid cross section.
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fig9: 3-D visualization of an ∼250-nm wide subunit spanning a fraction of the chromatid width supports a hierarchical folding model. (A) Dependence of labeled band width versus insert size reveals folding subunits. In a simple radial loop model (top), as the insert size increases, the labeled region spans an increasing fraction of the chromatid cross section, with the minimal width of a labeled band corresponding roughly to the diameter of a 30-nm chromatin fiber loop. With a successive coiling model, as the insert region increases in size, the labeled region spans an increasing fraction of the chromatid cross section, but the width of this labeled region does not increase until it spans a full chromatid cross section (bottom). The width of the minimal labeled region corresponds to the diameter of the folding subunit, significantly larger than a 30-nm chromatin loop. (B) Reconstructed orthogonal cross sections of EM serial section data built with NewVision. Arrows and arrowhead show the same nanogold-labeled areas as in Fig. 8, I–L. The spot-like labeled region appears as a labeled band extending across the chromatid in the orthogonal view. Thick red, green, and blue lines in images a, b, and c are, respectively, X, Y, and Z axes of a left orthogonal system with origin inside the nanogold-labeled insert. (C) Solid model display for same chromosome region shown in Fig. 8, I–L and Fig. 9 B reveals similar appearance of labeled regions on both chromatids. In the original serial sections, one region appeared as a band (Fig. 8 I, arrowhead) and one as a spot (Fig. 8, J and K, arrows). In the orthogonal views in B and in the solid model, both regions appear as ∼250–300-nm wide segments spanning only a fraction of the chromatid cross section.

Mentions: A distinct, ∼250-nm-diam coiling subunit within fully condensed metaphase chromosomes. A–D, E–H, and I–L show regions from individual serial sections from three different clone dSAR-d11 mitotic cells collected after nocodazole treatment, fixation, and immunogold labeling. Numbers represent positions of 40-nm thick sections in the original serial section stacks. Arrows and arrowhead label the two different labeled areas on the chromosome shown in I–L and displayed in three dimensions in Fig. 9, B and C. M–O show three independent examples of the small insert region from clone dSAR-g12. Bars: 0.2 μm for A–L, 0.3 μm for M–O.


Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

3-D visualization of an ∼250-nm wide subunit spanning a fraction of the chromatid width supports a hierarchical folding model. (A) Dependence of labeled band width versus insert size reveals folding subunits. In a simple radial loop model (top), as the insert size increases, the labeled region spans an increasing fraction of the chromatid cross section, with the minimal width of a labeled band corresponding roughly to the diameter of a 30-nm chromatin fiber loop. With a successive coiling model, as the insert region increases in size, the labeled region spans an increasing fraction of the chromatid cross section, but the width of this labeled region does not increase until it spans a full chromatid cross section (bottom). The width of the minimal labeled region corresponds to the diameter of the folding subunit, significantly larger than a 30-nm chromatin loop. (B) Reconstructed orthogonal cross sections of EM serial section data built with NewVision. Arrows and arrowhead show the same nanogold-labeled areas as in Fig. 8, I–L. The spot-like labeled region appears as a labeled band extending across the chromatid in the orthogonal view. Thick red, green, and blue lines in images a, b, and c are, respectively, X, Y, and Z axes of a left orthogonal system with origin inside the nanogold-labeled insert. (C) Solid model display for same chromosome region shown in Fig. 8, I–L and Fig. 9 B reveals similar appearance of labeled regions on both chromatids. In the original serial sections, one region appeared as a band (Fig. 8 I, arrowhead) and one as a spot (Fig. 8, J and K, arrows). In the orthogonal views in B and in the solid model, both regions appear as ∼250–300-nm wide segments spanning only a fraction of the chromatid cross section.
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fig9: 3-D visualization of an ∼250-nm wide subunit spanning a fraction of the chromatid width supports a hierarchical folding model. (A) Dependence of labeled band width versus insert size reveals folding subunits. In a simple radial loop model (top), as the insert size increases, the labeled region spans an increasing fraction of the chromatid cross section, with the minimal width of a labeled band corresponding roughly to the diameter of a 30-nm chromatin fiber loop. With a successive coiling model, as the insert region increases in size, the labeled region spans an increasing fraction of the chromatid cross section, but the width of this labeled region does not increase until it spans a full chromatid cross section (bottom). The width of the minimal labeled region corresponds to the diameter of the folding subunit, significantly larger than a 30-nm chromatin loop. (B) Reconstructed orthogonal cross sections of EM serial section data built with NewVision. Arrows and arrowhead show the same nanogold-labeled areas as in Fig. 8, I–L. The spot-like labeled region appears as a labeled band extending across the chromatid in the orthogonal view. Thick red, green, and blue lines in images a, b, and c are, respectively, X, Y, and Z axes of a left orthogonal system with origin inside the nanogold-labeled insert. (C) Solid model display for same chromosome region shown in Fig. 8, I–L and Fig. 9 B reveals similar appearance of labeled regions on both chromatids. In the original serial sections, one region appeared as a band (Fig. 8 I, arrowhead) and one as a spot (Fig. 8, J and K, arrows). In the orthogonal views in B and in the solid model, both regions appear as ∼250–300-nm wide segments spanning only a fraction of the chromatid cross section.
Mentions: A distinct, ∼250-nm-diam coiling subunit within fully condensed metaphase chromosomes. A–D, E–H, and I–L show regions from individual serial sections from three different clone dSAR-d11 mitotic cells collected after nocodazole treatment, fixation, and immunogold labeling. Numbers represent positions of 40-nm thick sections in the original serial section stacks. Arrows and arrowhead label the two different labeled areas on the chromosome shown in I–L and displayed in three dimensions in Fig. 9, B and C. M–O show three independent examples of the small insert region from clone dSAR-g12. Bars: 0.2 μm for A–L, 0.3 μm for M–O.

Bottom Line: We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models.Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes.Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Show MeSH
Related in: MedlinePlus