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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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Quantitation of scaffold association of vector sequence. Large inserts show enrichment of vector sequences over scaffold. (A) Extracted chromosome of clone dSAR-g12. Green, total DNA signal; red, vector DNA. (B) The size and shape of the scaffold depend on the threshold level. Colors are coordinated with the colored bar and threshold levels of C (arrowheads and arrows point to boundaries of two scaffolds defined by two intensity threshold levels shown in C). Higher threshold levels result in smaller scaffold regions. In C and D, the ordinate “scaffold enrichment ratio” represents the fraction of total operator signal localizing to the scaffold region divided by the fraction of total DNA localizing to the same region. Here, the scaffold region refers to the entire image region with intensity greater than the specific intensity threshold. Thus, the scaffold defined by arrows includes (but is larger than) the region defined by arrowheads. (C) Scaffold enrichment ratio versus threshold for the halo image shown in A. Intensity thresholds (vertical gray lines, marked by arrow or arrowhead) correspond to the actual scaffold regions (marked by arrows or arrowheads) shown in B. (D) Graphs represent the scaffold enrichment ratio versus threshold averaged over several halos; seven halos for Con-1 (con1), five halos for Con-610 (610), five and seven halos for smaller (d11-s) and larger (d11-l) inserts of clone dSAR-d11, respectively, and seven and four for smaller (g12-s) and larger (g12-l) inserts of clone dSAR-g12, respectively. Threshold values to the right of the vertical line correspond to defined scaffold regions that visually correspond to the DAPI core staining. Bar, 1 μm.
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fig4: Quantitation of scaffold association of vector sequence. Large inserts show enrichment of vector sequences over scaffold. (A) Extracted chromosome of clone dSAR-g12. Green, total DNA signal; red, vector DNA. (B) The size and shape of the scaffold depend on the threshold level. Colors are coordinated with the colored bar and threshold levels of C (arrowheads and arrows point to boundaries of two scaffolds defined by two intensity threshold levels shown in C). Higher threshold levels result in smaller scaffold regions. In C and D, the ordinate “scaffold enrichment ratio” represents the fraction of total operator signal localizing to the scaffold region divided by the fraction of total DNA localizing to the same region. Here, the scaffold region refers to the entire image region with intensity greater than the specific intensity threshold. Thus, the scaffold defined by arrows includes (but is larger than) the region defined by arrowheads. (C) Scaffold enrichment ratio versus threshold for the halo image shown in A. Intensity thresholds (vertical gray lines, marked by arrow or arrowhead) correspond to the actual scaffold regions (marked by arrows or arrowheads) shown in B. (D) Graphs represent the scaffold enrichment ratio versus threshold averaged over several halos; seven halos for Con-1 (con1), five halos for Con-610 (610), five and seven halos for smaller (d11-s) and larger (d11-l) inserts of clone dSAR-d11, respectively, and seven and four for smaller (g12-s) and larger (g12-l) inserts of clone dSAR-g12, respectively. Threshold values to the right of the vertical line correspond to defined scaffold regions that visually correspond to the DAPI core staining. Bar, 1 μm.

Mentions: The real question in analyzing these images is whether the apparent association (or lack of association) with the scaffold region is meaningful; ideally, we need to normalize the observed DNA distribution to test whether the vector sequence is distributed between halo and scaffold quantitatively different than the total DNA. We wish to measure the fold enrichment of vector sequence relative to total DNA present in the scaffold fraction. This parallels most closely typical biochemical analysis of sequence fractionation in the scaffold fraction, where relative distribution of putative SAR sequences are measured between equal amounts of scaffold versus halo (supernatant) DNA loaded per sample. This “scaffold enrichment ratio” was measured by normalizing the operator signal by the DNA signal present in the same compartment. Specifically, we computed the ratio of the fraction of total operator signal localizing to the scaffold region divided by the fraction of DAPI staining localizing to the same scaffold region. We defined the scaffold region as corresponding to the collection of all pixels whose DAPI intensity exceeded a given threshold. Because the scaffold perceived visually did not have an exact boundary (Fig. 4, A and B)Figure 4.


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)

Quantitation of scaffold association of vector sequence. Large inserts show enrichment of vector sequences over scaffold. (A) Extracted chromosome of clone dSAR-g12. Green, total DNA signal; red, vector DNA. (B) The size and shape of the scaffold depend on the threshold level. Colors are coordinated with the colored bar and threshold levels of C (arrowheads and arrows point to boundaries of two scaffolds defined by two intensity threshold levels shown in C). Higher threshold levels result in smaller scaffold regions. In C and D, the ordinate “scaffold enrichment ratio” represents the fraction of total operator signal localizing to the scaffold region divided by the fraction of total DNA localizing to the same region. Here, the scaffold region refers to the entire image region with intensity greater than the specific intensity threshold. Thus, the scaffold defined by arrows includes (but is larger than) the region defined by arrowheads. (C) Scaffold enrichment ratio versus threshold for the halo image shown in A. Intensity thresholds (vertical gray lines, marked by arrow or arrowhead) correspond to the actual scaffold regions (marked by arrows or arrowheads) shown in B. (D) Graphs represent the scaffold enrichment ratio versus threshold averaged over several halos; seven halos for Con-1 (con1), five halos for Con-610 (610), five and seven halos for smaller (d11-s) and larger (d11-l) inserts of clone dSAR-d11, respectively, and seven and four for smaller (g12-s) and larger (g12-l) inserts of clone dSAR-g12, respectively. Threshold values to the right of the vertical line correspond to defined scaffold regions that visually correspond to the DAPI core staining. Bar, 1 μm.
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Related In: Results  -  Collection

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fig4: Quantitation of scaffold association of vector sequence. Large inserts show enrichment of vector sequences over scaffold. (A) Extracted chromosome of clone dSAR-g12. Green, total DNA signal; red, vector DNA. (B) The size and shape of the scaffold depend on the threshold level. Colors are coordinated with the colored bar and threshold levels of C (arrowheads and arrows point to boundaries of two scaffolds defined by two intensity threshold levels shown in C). Higher threshold levels result in smaller scaffold regions. In C and D, the ordinate “scaffold enrichment ratio” represents the fraction of total operator signal localizing to the scaffold region divided by the fraction of total DNA localizing to the same region. Here, the scaffold region refers to the entire image region with intensity greater than the specific intensity threshold. Thus, the scaffold defined by arrows includes (but is larger than) the region defined by arrowheads. (C) Scaffold enrichment ratio versus threshold for the halo image shown in A. Intensity thresholds (vertical gray lines, marked by arrow or arrowhead) correspond to the actual scaffold regions (marked by arrows or arrowheads) shown in B. (D) Graphs represent the scaffold enrichment ratio versus threshold averaged over several halos; seven halos for Con-1 (con1), five halos for Con-610 (610), five and seven halos for smaller (d11-s) and larger (d11-l) inserts of clone dSAR-d11, respectively, and seven and four for smaller (g12-s) and larger (g12-l) inserts of clone dSAR-g12, respectively. Threshold values to the right of the vertical line correspond to defined scaffold regions that visually correspond to the DAPI core staining. Bar, 1 μm.
Mentions: The real question in analyzing these images is whether the apparent association (or lack of association) with the scaffold region is meaningful; ideally, we need to normalize the observed DNA distribution to test whether the vector sequence is distributed between halo and scaffold quantitatively different than the total DNA. We wish to measure the fold enrichment of vector sequence relative to total DNA present in the scaffold fraction. This parallels most closely typical biochemical analysis of sequence fractionation in the scaffold fraction, where relative distribution of putative SAR sequences are measured between equal amounts of scaffold versus halo (supernatant) DNA loaded per sample. This “scaffold enrichment ratio” was measured by normalizing the operator signal by the DNA signal present in the same compartment. Specifically, we computed the ratio of the fraction of total operator signal localizing to the scaffold region divided by the fraction of DAPI staining localizing to the same scaffold region. We defined the scaffold region as corresponding to the collection of all pixels whose DAPI intensity exceeded a given threshold. Because the scaffold perceived visually did not have an exact boundary (Fig. 4, A and B)Figure 4.

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