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. 2008 May 19;181(4):615-23.
doi: 10.1083/jcb.200710053.

Similar active genes cluster in specialized transcription factories

Meng Xu  1 , Peter R Cook
Affiliations

Similar active genes cluster in specialized transcription factories

Meng Xu et al. J Cell Biol. .

Abstract

How transcription affects the way specific genes are arranged within the nucleus remains to be fully understood. We examine here whether transcription occurs in discrete sites (factories) containing the required machinery and whether these sites specialize in transcribing different genes. We cotransfected plasmids encoding a common origin of replication but different transcription units into cells, where they are assembled into minichromosomes that the cellular machinery replicates and transcribes. In cells containing thousands of minichromosomes, we found (using fluorescence in situ hybridization) active templates concentrated in only a few factories that transcribe particular units depending on the promoter type and the presence of an intron. Close proximity between similar transcription units, whether on two different minichromosomes or on host chromosomes and minichromosomes, is confirmed using chromosome conformation capture. We conclude that factories specialize in producing a particular type of transcript depending on promoter type and whether or not the gene contains an intron.

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Figures

Figure 1.
Figure 1.
Approach. (A) Plasmids were cotransfected into cells, where they replicate, and we asked: Are the resulting minichromosomes spread throughout nuclei, or targeted to the same or different factories? (B) Different promoters, coding regions, introns, and 3′ ends (gray regions) are inserted into a common backbone (with SV40 ori + early promoter driving Neor).
Figure 2.
Figure 2.
Replicating minichromosomes are concentrated in few foci. Cells were cotransfected with IICMV,DsRed,pA (so transfection efficiencies could be monitored by FACS using DsRed fluorescence) and another encoding EGFP as indicated; after 8 h, cells were replated (to wash away input) and regrown for 4, 16, and 28 h. (A) The DNA copy number of IICMV,EGFP,pA per transfected cell—determined by reference to known amounts of pure plasmid DNA (by blotting using a “Hirt” extract and qPCR using total DNA)—increases above the maximum possible background due to input (upper limit of gray area). Numbers (24 h) obtained in analogous experiments for I45S,EGFP,pA, III7SK,EGFP,pT, and IICMV,EGFPV,pA were similar (i.e., 23,000 ± 2,000, 18,000 ± 3,000, and 21,000 ± 3,000, respectively; one-way ANOVA, P > 0.05). (B) Promoters and introns affect the RNA copy number per transfected cell (determined using qRT-PCR by reference to known amounts of pure RNA). (C) DNA FISH shows that minichromosomes are concentrated in ∼20 foci. (inset) Fluorescent bead used for normalization. Bar, 2.5 μm. (D) Mean intensities (+SD) of pixels in or outside foci in 100 images like those in C were normalized relative to the beads; most signal is in the foci, and this increases with time.
Figure 3.
Figure 3.
RNA FISH shows nascent minichromosomal RNA in few nuclear foci. Cells were transfected and grown for 24 h, minichromosomal transcripts were detected by RNA FISH, DNA was counterstained with DAPI, and images were collected. (A) Two sets of four views of one field are shown. (bottom) Percentages (± SD) of green foci that overlap red foci, and vice versa. (i–iv) Transfection with IICMV,DsRed,pA. An untransfected cell (arrowhead) contains no DsRed or Neor transcripts. The other contains many cytoplasmic DsRed transcripts but few Neor transcripts; its nucleus contains some red and green foci (marking nascent RNA at transcription sites) against a general background (marking transcripts on their way to the cytoplasm). (insets) Two foci with both red and green fluorescence; this is expected, as the plasmid encodes both DsRed and Neor (on the backbone). (v–viii) Cotransfection with IICMV,DsRed,pA and IICMV,EGFP,pA, which differ solely in coding region. The two central (transfected) cells contain DsRed and EGFP RNA mainly in the cytoplasm, with some in the nuclear foci. (insets) Nuclear focus with both types of RNA. Insets show an enlarged view of the boxed portions. (B) Discriminating between nuclear foci and background. Intensities are expressed relative to those given by fluorescent reference beads, and the fraction of foci in 200 cells with relative intensities of 0–0.05, 0.06–0.1, etc., is indicated. Promoterless 0,EGFP,pA gives faint EGFP signal due to autofluorescence (equivalent to that seen in mock-transfected cells, not depicted; gray bar) and read-through from the SV40 early promoter into EGFP (red bars). For plasmids with promoters (e.g., IICMV,EGFP,pA), only foci with intensities greater than the maximum read-through (green dotted line) were considered. (C) Cells were transfected with IICMV,EGFP,pA and incubated with or without α-amanitin and RNase, DNA was stained with DAPI, and EGFP transcripts were detected; the treatments abolish EGFP signal. Three sets of two views of one field are shown. Bars: (A) 5 μm; (C) 10 μm.
Figure 4.
Figure 4.
Promoters and introns target minichromosomes to specific factories. Cells (cotransfected with IICMV,DsRed,pA and the plasmid indicated) were grown (24 h), transcripts were detected by RNA FISH, and DNA was counterstained with DAPI. Four views of one field for each pair are shown. (bottom) Percentages (± SD) of green foci that overlap red foci, and vice versa. Arrows indicate noncolocalizing foci and arrowheads indicate colocalizing foci. Insets show an enlarged view of the boxed portions. (A–D) Different polymerase II units. One transfected cell contains DsRed but not U2G transcripts in the cytoplasm; both are found in nuclear foci. (insets) Nuclear foci do not colocalize (confirmed by low percentages shown on the bottom). (E–H). Plus/minus intron. Both cells contain DsRed and EGFP transcripts in cytoplasm and nucleus. (insets) The top focus contains both types, whereas the bottom shows only EGFP RNA (infrequent colocalization is reflected by low percentages on the bottom). (I–L) I + II. The central transfected cell contains DsRed but not EGFP RNA in the cytoplasm; nuclear foci do not colocalize. (insets) Foci do not colocalize (as confirmed by the low percentages shown below). (M–P) II + III. The transfected cell (left) contains both types of RNA in cytoplasm and nucleus. (insets) Foci do not colocalize (as confirmed by the low percentages shown below). Bar, 5 μm.
Figure 5.
Figure 5.
ChIP-3C shows that similar genes on mini- and host chromosomes lie together. Cells were transfected, grown for 24 (rows 1–7) or 8 h (rows 8–13), and treated with or without cross-linker. Active and inactive chromatin was separated by ChIP (using an antibody against trimethyl K4 in H3) and the proximity between selected genes was assessed by 3C. Images illustrate gels containing amplimers, whereas rows 7 and 13 give the relative cross-linking frequency (the amounts of 3C amplimers in bands were determined by reference to equivalent weights of DNA and normalized relative to ALDOA–ALDOA levels and the PCR control for amplification efficiency). ALDOA is an active housekeeping gene on the host chromosome. (A) Minichromosome–minichromosome. Cotransfection with IICMV,DsRed,pA + IICMV,EGFP,pA (II + II) or IICMV,DsRed,pA + III7SK,EGFP,pT (II + III). DsRed–EGFP 3C products were obtained from both supernatant and pellet (rows 1 and 3), which is consistent with naked input and/or active minichromosomes in the supernatant, and they were seen only after cross-linking (X-link; rows 1 and 4). More are seen in the pellet when promoters are identical (row 3, compare left and right lanes; row 7, mean of four experiments); therefore, templates lie closer together in the II + II combination than in the II + III combination. ALDOA–ALDOA 3C products are seen only in the pellet (rows 2 and 5) and in similar amounts in both combinations (row 5). Amplimers are obtained in roughly equal amounts using the same primers and synthetic 3C templates made by ligating equimolar amounts of pure DNA from relevant genes (row 6). (B) Minichromosome–host chromosome. Transfection with IIU2,U2G,3′ box (IIU2) or IICMV,EGFP,pA (IICMV). More plasmid–host 3C products (detected using primers targeting the plasmid backbone and sequences flanking host U2 genes) are seen with IIU2 than with IICMV (row 1, compare left lane with the right; row 6, mean of four experiments); therefore, IIU2 lies closer to host U2 units than IICMV. In contrast, amplimers from ALDOA–ALDOA, synthetic 3C templates, and the Neor gene on the backbone (which reflect plasmid copy number) are all obtained in roughly equal amounts (rows 3–5).
Figure 6.
Figure 6.
Models. (A) Targeting to factories. The promoter encoded by the minichromosome in the center can initiate only in a factory containing the appropriate machinery (i.e., one of similar color). As a result, it forms a cluster with two similar minichromosomes by association with the factory on the right. (B) Intron targeting. A newly replicated intron-containing minichromosome is “naïve” (left), and may either bind and initiate (incorrectly) in a nonsplicing (polymerase II) factory (bottom, blue) or a “splicing” factory (top, magenta), where it acquires a mark during splicing (e.g., a histone modification; right) that now targets it to the appropriate (splicing) factory. (C) Chromosome pairing. Just as similar (“homologous”) minichromosomes cluster (“pair”) in A, a similar process may underlie the pairing of homologous chromosomes in both somatic and meiotic cells. For example, during meiosis, homologues seek out and align with their partners before the close synapsis that occurs during recombination. It is now accepted that distinct mechanisms underlie alignment and synapsis, as homologues still align in mutants unable to carry out the later steps (McKee, 2004; Gerton, and Hawley, 2005). During alignment, homologues are transcriptionally active (Cook, 1997) so that each chromosome in the haploid set will possess a unique array of active transcription units running from telomere to telomere. Only the homologue will possess a similar array. Here, only one of the many loops associated with a factory is shown. The yellow promoter on maternal chromosome 2 (2m) is unlikely to bind to the green factory on maternal chromosome 1 (1m). But just as a factory of a particular type nucleates pairing between minichromosomes bearing similar transcription units, correct alignment begins when the yellow (2m) promoter binds to the yellow factory on its homologue (2p). Once transcription of the yellow promoter on 2m begins, 2m and 2p become temporarily tethered together, and this will increase the chances that adjacent promoters bind to homologous factories (i.e, the gray unit on 2m with the gray factory on 2p, etc.). As a result, 2m and 2p eventually become zipped together (arrowhead) and thus aligned.
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