Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos
UMass Chan Affiliations
Department of Cell BiologyDocument Type
Journal ArticlePublication Date
1994-07-01Keywords
Cell CycleCell Line
Cell Line, Transformed
Cell Nucleus
Centromere
Chromatin
Chromosomes, Human
DNA
DNA Probes
DNA Replication
Fibroblasts
Hela Cells
Humans
In Situ Hybridization, Fluorescence
Multigene Family
*Nucleic Acid Conformation
Protein Conformation
RNA, Small Nuclear
Repetitive Sequences, Nucleic Acid
X Chromosome
Cell Biology
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.Source
J Cell Biol. 1994 Jul;126(2):289-304. Link to article on publisher's websiteDOI
10.1083/jcb.126.2.289Permanent Link to this Item
http://hdl.handle.net/20.500.14038/42623PubMed ID
8034736Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1083/jcb.126.2.289