Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods

Abstract

Typically, eukaryotic nuclei contain 10-30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain "choice" appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3-4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments ( approximately 5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic "neighborhoods."

Figure 1.

COL1A1 and COL1A2 genes associate with a single, common domain. (A) WI-38 diploid fibroblasts were hybridized with differently labeled genomic probes of COL1A1 (red) and COL1A2 gene (green) and stained for SC-35 (blue). One homologue of each gene is simultaneously associated with the same SC-35 domain in the cell shown. (B) Transcripts from the COL1A1 (green) and COL1A2 (red) genes, detected with differentially labeled cDNA probes, intermingle within an SC-35 domain (blue). Overlap between the three colors appears white. (C) Three-dimensional deconvolution shows intermingling COL1A1 (green) and COL1A2 (red) transcripts in two focal planes. Regions of colocalization appear yellow. To view a three-dimensional reconstruction of this stack, see supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200303131/DC1). Bars, 5 μm.

Figure 2.

Transcripts from multiple genes can associate with the same SC-35 domain. (A) One focus of lamin A/C RNA (red) associates with the edge of an accumulation of COL1A1 transcripts (green), which serves as a marker for an SC-35 domain. (B) Triple labeling also shows that transcripts from ACTB (green) and COL1A2 (red) accumulate within the same SC-35 domain (blue). (C) Chromosome 7 territories detected with a whole chromosome paint (red) contact 3–4 SC-35 domains (green) per nucleus. Bars, 5 μm.

Figure 3.

Specificity of chromosome band probes. Probes from indicated bands were hybridized to spreads of human metaphase chromosomes (stained with DAPI, blue) of peripheral blood lymphocytes to assess their specificity. (A) 7p21, 17q21 (both green), and 17q22–24 (red); (B) 3p14 (red), 7q21 (green), and 19q13.3 (green); (C) 6p21.3 (red). Probes for 7p21 and 7q21 weakly detected additional bands, but 7p21 DNA was identified in interphase based on signal size, intensity, and cohybridization of the neighboring ACTB locus. Signals from 7q21 probe were not evaluated in interphase nuclei. (D–J) Enlarged views of single chromosomes, shown to scale, hybridized with different band probes as indicated and compared with DAPI bands (blue or white) indicates that they are largely either R- or G- band DNA. In D, the COL1A1 gene (red) is cohybridized with 17q21 (green), showing that this gene maps to the telomeric end of this band, proximal to 17q22–24 (E). Bars, 5 μm.

Figure 4.

Differential distribution of R- and G-band DNA with respect to SC-35 domains in interphase nuclei. (A and B) Probe of R-band 17q21 DNA (red in A, white in B) hybridized to WI-38 fibroblasts shows two highly extended homologues, each with different morphologies. The left homologue contacts four SC-35 domains (green). The right band surrounds half of one SC-35 domain and also contacts another. (C and D) Two 6p21.3 R-bands (red, white) each have a thin string of DNA extending from the more compact, main body of the band. In one case (top band), the extended region almost completely surrounds an SC-35 domain (green). (E) A tetraploid nucleus hybridized to detect 17q21 DNA (green) shows all four bands contacting multiple SC-35 domains (blue). The COL1A1 gene (red, right inset) is located at the tip of a DNA extension that contacts an SC-35 domain at a single point, in contrast to another homologue (left inset). (F and G) The G-band probe for 3p14 (red in F, white in G) shows two bands minimally contacting SC-35 domains (green). (H and I) Similar to other G-bands, probe for 7p21 (red, white) was detected in focal planes other than those containing SC-35 domains (green), which thus appear out of focus. (J) Hybridization to detect 17q22–24 DNA (red) and the adjacent COL1A1 gene (green) shows that although some of these bands can extensively contact the SC-35 domain (blue) associated with COL1A1 (top inset), others are clearly separated (bottom inset). (K) Three-dimensional deconvolution and reconstruction of 17q21 R-band signal (red) and surrounding SC-35 domains (green) shows that this band extends significantly in the X-Y plane (left), but when viewed along the Z-axis (right), appears largely restricted to the planes containing SC-35 domains. (L) A three-dimensional reconstruction as in K shows an example of a G-band, 3p14 (red), located above and separated from the nearest SC-35 domain (green). For rotational movies of K and L, see supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200303131/DC1). Bars, 5 μm.

Figure 5.

Differential organization of early- and later-replicating DNA relative to SC-35 domains. (A) An early S-phase nucleus pulse labeled with BrdU (green) shows the typical pattern of hundreds of small early-replication foci throughout the nuclear interior, excluding the nucleolus. All SC-35 domains (red) are contacted by multiple early-replicating foci, which correspond to gene-rich DNA. Nuclei in mid (B) and late (C) S-phase are identified by replication sites of gene-poor DNA at the nuclear periphery or in large clumps that are more interior but on different focal planes than SC-35 domains. Arrows indicate SC-35 domains enlarged in insets.

Figure 6.

Data presented here support model 2 (center) rather than model 1 (left), and are further summarized at far right. (Model 1) mRNA metabolic factors, including SC-35 (green), accumulate on transcripts (yellow) of a single highly active gene (red, top left) or genomic clusters of genes (bottom left). This model requires no structural organization of genes relative to the large concentrations of splicing factors, which merely reflect the distribution of transcripts on different genes. (Model 2) Multiple genes (center top) cluster at the periphery of a single large accumulation of mRNA metabolic factors. R-band DNA (light blue), which is gene rich, is more intimately associated with these SC-35 domains than gene-poor G-band DNA (dark blue, bottom center). (Right) Each chromosome territory (aqua) associates with three or four SC-35 domains, indicating specialized regions at the chromosome territory periphery. These contain domain-associated genes that can even come from different chromosome arms. An SC-35 domain can also associate with genes from different chromosomes. Because domain choice for individual genes is often random, the relative positions of their respective chromosomes also may be highly variable.