Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction

Abstract

Specific chromosome domains in interphase nuclei of neurons and glia were studied by three-dimensional (3-D) reconstruction of serial optical sections from in situ hybridized human CNS tissue. Overall patterns of centromere organization, delineated with alphoid repeats, were comparable to those seen in mouse, and are clearly conserved in mammalian evolution. Cloned probes from other individual chromosome domains were used to define interphase organization more precisely. Homologous chromosomes were spatially separated in nuclei. In large neurons, probes specific for 9q12, or 1q12 showed that at least one homolog was always compartmentalized together with centromeres on the nucleolus, while the second signal either abutted the nucleolus or was on the nuclear membrane. A telomeric Yq12 sequence also localized together with perinucleolar centromeres in a completely non-Rabl orientation. In astrocytes, these three chromosome regions were on the membrane and not necessarily associated with nucleoli. Therefore there are different patterns of interphase chromosome organization in functionally distinct cell types. In contrast to the above domains, a 1p36.3 telomeric sequence embedded in a large Alu-rich and early replicating chromosome region, was always found in an interior euchromatic nuclear compartment in both neurons and glial cells. In double hybridizations with 1q12 and 1p36.3 probes, 1p arms were clearly separated in all cells, and arms projected radially into the interior nucleoplasm with non-Rabl orientations. There was no absolute or rigid position for each 1p arm with respect to each other or to the major dendrite, indicating that individual chromosome arms may be dynamically positioned even in highly differentiated cell types. We suggest that centromeric and other highly repeated non-transcribed sequence domains may act as general organizing centers for cell type specific interphase patterns that are conserved in mammalian evolution. Such centers would allow selected groups of chromosome arms to extend into (and contract from) an interior, presumably transcriptionally active, nuclear compartment.

A unified model of eukaryotic chromosomes

“…This molecular segregation of LINE and SINE motifs into structurally distinct compartments has recently been confirmed (30). In extended interphase chromosomes, LINE repeats are detected as -250 nm wide spots of varying length (46,47). From the structural model above it can be predicted that linear DNAs of 300 kb to >1.5Mb should contain clusters of LINE sequences.…”

“…In contrast, early repli- Urd-peroxidase labeled) in prometaphase spread. Open arrow points to labeled fine domain a t 1~36.3, likely to be the site of a minisatellite (47); the surrounding extended early replicating regions that are unlabeled have previously been shown to contain a high concentration of GC rich SINES (46). The length of chromosome 1 in this prometaphase preparation (that is not maximally extended) is 10 Fm.…”

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

“…Several lines of evidence suggest that the perinucleolar compartment functions as a repressive environment in other cell types. Nucleoli in higher eukaryotes are surrounded by a layer of heterochromatin (1,4,5,(48)(49)(50). In mammalian ES cell cultures, the inactive X chromosome (Xi) or autosomes expressing the Xist transcript visit the perinucleolar space in S phase (9).…”

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

“…In particular, centromeric and other highly repeated nontranscribed sequence domains may be positioned on the nucleolus or nuclear membrane and act as general organizing centers for cell type-specific interphase patterns (21). Multiple adjacent replicons appear to be clustered on fixed sites of the nuclear matrix (17,271, and it has been suggested that clustered replication origins are activated simultaneously (25).…”