Studies of Metaphase and Interphase Chromosomes Using Fluorescence In Situ Hybridization

Trask, Barbara J.; Allen, Sally Lyman; Massa, Hillary F.; Fertitta, Anne; Sachs, Rainer K.; Engh, Ger van den; Wu, Madeline

doi:10.1101/sqb.1993.058.01.084

Cited by 72 publications

(38 citation statements)

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“…These latter determinations are performed at low ionic strengths in vitro, which are conditions that tend to unfold chromatin. Although our value is somewhat higher than that estimated from formaldehyde crosslinking studies of Chr3 (16), it is consistent with the values predicted from simulations and FISH measurements in human fibroblast nuclei (5,14,33). Our study predicts Lp based on measurements over small intervals monitored under physiological conditions.…”

Section: Discussionsupporting

confidence: 79%

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

Bystricky

Heun

Gehlen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

288

330

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Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14 -100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170 -220 nm and a mass density of Ϸ110 -150 bp͞nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.higher-order structure ͉ 30-nm fiber ͉ nucleosomes G enetic studies indicate that the spatial positioning of the genome in interphase contributes to the regulation of nuclear functions, yet the principles that govern the organization of interphase chromosomes (Chrs) are largely unknown. At the simplest level, DNA is folded through interaction with histones forming the nucleosome core particle (NCP), which yields a 6:1 or 7:1 compaction ratio depending on linker length. Arrays of nucleosomes are further condensed by Ϸ6-fold into a higherorder structure, the so-called 30-nm fiber, whose in vivo architecture is unresolved. Several models have been proposed for this structure (ref. 1 and reviewed in ref. 2), yet species' specific variation in linker histones and nucleosome repeat lengths may lead to variation in fiber characteristics. How interphase Chrs fold beyond this level of organization is even less well understood, although in addition to fiber compaction, local looping and͞or anchoring to subnuclear elements may influence chromatin conformation (3, 4).To analyze chromatin compaction ratios in interphase nuclei, laboratories have generally applied fluorescence in situ hybridization (FISH), using differentially derivatized probes. These data were interpreted as identifying mega bp-sized loops (averaging 3,000 kb) with the bases of the loops being distributed in a random walk throughout the nucleoplasm (5-7) or as a chain of chromosomal subcompartments each comprising Ϸ10-20 loops of Ϸ120 kb (8). The random distribution of the chain may reflect local chromatin dynamics, which have been recently well documented in living cells by rapi...

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Section: Discussionsupporting

confidence: 79%

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

Bystricky

Heun

Gehlen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

288

330

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“…An abrupt change in the polymer-like behavior of interphase chromatin was observed around 2 Mbp (Trask et al, 1991(Trask et al, , 1993avan den Engh et al, 1992). Above this genomic separation, mean interphase distance appeared to plateau.…”

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confidence: 99%

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

Yokota

Engh

Hearst

et al. 1995

The Journal of Cell Biology

271

200

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Abstract. We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at N2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales >100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation. SIaORXLY after cell division, the mitotic chromosomes decondense and diffuse into the interphase nucleus. While individual chromosomes cannot be discerned, important processes related to chromosome function take place. Regulatory factors interact with chromatin, DNA is made accessible for transcription, RNA is produced and processed, DNA is replicated, and repairs are made of DNA strand breaks. When the chromosomes reappear for the next mitosis, they have been duplicated and prepared for rapid partitioning over the daughter cells. The complexity of these processes raises many questions about the large-scale organization of chromosomes and how this organization relates to cell function (e.g., Blobel, 1985;Manuelidis and Chen, 1990;Cook, 1991;Lawrence and Singer, 1991;De Boni, 1994).Diverse models, ranging from highly random to highly organized, have been proposed for the higher-order organization of interphase chromatin. These models variously involve irregularly folded fibers (DuPraw, 1965), radial loop structures (Manuelidis and Chen, 1990), giant loops (Ostashevsky and Lange, 1994), semirigid orientation ("Rabl" configuration) (Rabl, 1885;Comings, 1968), or random polymers confined by tethering or external forces (Hahnfeldt et al., 1993). Some models assign to chromo-

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“…Between 50 and 100 interphase distances were measured for each telomere. Genomic distance was estimated from a calibration curve that was produced in our laboratory using the same cosmids used to create the published relationship between the mean interphase distance squared and the genomic distance for the chromosomal region 4p16.3 (Trask et al 1993).…”

Section: Interphase Distance Measurementsmentioning

confidence: 99%

Characterization of Physical Gap Sizes at Human Telomeres

Lese¹,

Fantes²,

Riethman³

et al. 1999

Genome Res.

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Genome-wide physical and genetic mapping efforts have not yet fully addressed the problem of closure at the telomeric ends of human chromosomes. Targeted efforts at cloning human and mouse telomeres have succeeded in identifying unique sequences at most telomeres, but gap sizes between these telomere clones and the distal markers on integrated genetic/physical maps remain largely unknown. As telomeric regions are known to be the most gene-rich regions of the human genome, filling these gaps should have a high priority in completion of the Human Genome Project. We reported previously a first generation set of unique sequence probes for human telomeric regions. Of 41 human telomere regions, 33 were represented by unique clones with a known distance (Յ300 kb) from the end of the chromosome; clones for the remaining eight telomeric regions had not yet been identified and were represented by the most distal markers on the integrated genetic/physical map. We have identified unique telomere clones for four of the remaining telomeres, 9p, 12p, 15q, and 16p. To determine the telomeric gap size for these chromosomes and five other human telomeres, interphase FISH analysis was performed to measure the distance between each telomere clone and the corresponding most distal marker. These studies provide distance estimates ranging from <100 kb to >1 Mb, thus defining the physical mapping task for filling telomeric gaps.

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Studies of Metaphase and Interphase Chromosomes Using Fluorescence In Situ Hybridization

Cited by 72 publications

References 0 publications

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

Characterization of Physical Gap Sizes at Human Telomeres

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