Analysis of human interphase chromosome territories in vivo

Chromosome territories need to be well defined at high resolution before functional aspects of chromosome organization in interphase can be explored. To visualize chromosomes by electron microscopy (EM), the DNA of Chinese hamster fibroblasts was labeled in vivo with thymidine analogue BrdU. Labeled chromosomes were then segregated during several cell cycles to obtain nuclei containing only 2 to 3 labeled chromosomes. Subsequent immunocytochemical detection of BrdU allowed analysis by EM of chromosome territories and subchromosomal domains in well preserved nuclei. Our results provide the first high resolution visualization of chromosomes in interphase nuclei. We show that chromosome domains are either separated from one another by interchromatin space or are in close contact with no or little intermingling of their DNA. This demonstrates that, while chromosomes form discrete territories, chromatin of adjacent chromosomes may be in contact in limited regions, thus implying chromosome-chromosome interactions. Chromosomes are organized as condensed chromatin with dispersed chromatin extending into the interchromatin space that is largely devoid of DNA. The interchromatin space, which is known to be involved in various nuclear functions, forms interconnecting channels running through and around chromosome territories. Functional implications of this organization are discussed.

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Structure and dynamics of human interphase chromosome territories in vivo

Human Genetics ◽

10.1007/s004390050686 ◽

1998 ◽

Vol 102 (2) ◽

pp. 241-251 ◽

Cited By ~ 216

Author(s):

D. Zink ◽

T. Cremer ◽

Rainer Saffrich ◽

Roger Fischer ◽

Michael F. Trendelenburg ◽

...

Keyword(s):

Chromosome Territories ◽

Interphase Chromosome ◽

Structure And Dynamics

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Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template

The Journal of Cell Biology ◽

10.1083/jcb.200809196 ◽

2009 ◽

Vol 185 (1) ◽

pp. 87-100 ◽

Cited By ~ 80

Author(s):

Yan Hu ◽

Igor Kireev ◽

Matt Plutz ◽

Nazanin Ashourian ◽

Andrew S. Belmont

Keyword(s):

Chromatin Structure ◽

Transcriptional Activation ◽

Large Scale ◽

Nuclear Speckles ◽

Interphase Chromosome ◽

Chromosome Conformation ◽

Dynamic Events ◽

Classical Models ◽

Chromatin Fibers

The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.

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Labeling DNA Replication Foci to Visualize Chromosome Territories In Vivo

Current Protocols in Cell Biology ◽

10.1002/cpcb.19 ◽

2017 ◽

Vol 75 (1) ◽

Cited By ~ 1

Author(s):

Apolinar Maya‐Mendoza ◽

Dean A. Jackson

Keyword(s):

Dna Replication ◽

Chromosome Territories ◽

Replication Foci

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Spatial Relationship between Transcription Sites and Chromosome Territories

The Journal of Cell Biology ◽

10.1083/jcb.147.1.13 ◽

1999 ◽

Vol 147 (1) ◽

pp. 13-24 ◽

Cited By ~ 162

Author(s):

Pernette J. Verschure ◽

Ineke van der Kraan ◽

Erik M.M. Manders ◽

Roel van Driel

Keyword(s):

X Chromosome ◽

Large Scale ◽

Fluorescent Protein ◽

Spatial Relationship ◽

Chromosome Territory ◽

Chromosome Territories ◽

Interphase Chromosome ◽

Chromatin Domains ◽

Transcription Sites ◽

Nascent Rna

We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300–450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

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