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First published online 29 August 2006
doi: 10.1242/jcs.03050

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Homologous gene sequences mediate transcription-domain formation

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

View larger version (64K): [in a new window]   Fig. 1. Transiently transfected plasmids are transcribed in TDs or in a punctate pattern. (A) Plasmid maps of pHIV-ß, pHIV- and pHIV-ß. Boxes indicate promoters and exons, black arrows indicate transcriptional start sites, and `pA' indicates polyadenylation sites. The plasmids are based on the pUC18 backbone (not shown) and contain the HIV-LTR promoter upstream of the plasmid gene. (B) S1 nuclease analysis of cytoplasmic RNA from HeLa cells transiently transfected with pHIV-ß, pHIV-ß or pCMV-ß. The labelled probe was complementary to the third exon and 3' flank sequence of ß-globin, and mismatched with correctly processed mRNAs at the polyadenylation site. All plasmids generated correctly processed mRNAs corresponding to the band marked `ßpA' in lanes 1, 3 and 4. Transcription of pHIV-ß was Tat-dependent (compare lanes 2 and 3), whereas transcription of pCMV-ß was Tat-independent (lane 1). pHIV-ß was also co-transfected with plasmid pHIV-, which had no discernible effect on transcription levels (compare lanes 3 and 5). (C) RNA in situ hybridisation of HeLa cells transiently transfected with pHIV-ß. The majority of transfected nuclei displayed five to 20 discrete nascent RNA signals corresponding to plasmid TDs (left panel). A minority of cells displayed a punctate pattern of plasmid transcription in which many transcription sites were scattered throughout the nucleoplasm (right panel). Bars, 5 µm.

View larger version (92K): [in a new window]   Fig. 2. (A) TDs and punctate plasmid transcription sites represent nascent transcription. HeLa cells were transiently transfected with pHIV-ß and plasmid transcription was double-labelled by RNA in situ hybridisation using probes to the ß-globin first or second intron (left panel) and 3' flank (middle panel). The RNA signals colocalised, as indicated by the yellow colour in the overlay images (right panel). Bars, 1 µm. (B) Plasmid TDs localise adjacent to the interchromatin granule clusters (IGCs) and the PML bodies. HeLa cells were transiently transfected with pHIV-ß and plasmid transcription sites were labelled by RNA in situ hybridisation using probes to the ß-globin first intron (green signals). IGCs were labelled with an anti-SC35 antibody (top panels) and PML bodies were labelled with an anti-PML antibody (bottom panels). TDs were consistently observed adjacent to both IGCs and PML bodies. Bars, 5 µm.

View larger version (91K): [in a new window]   Fig. 3. Plasmid TDs localise to the interchromatin compartment, whereas punctate plasmid transcription sites overlie the cellular chromatin. (A) Plasmid pHIV-ß was transiently transfected into HeLa cells expressing a GFP-histone H2B construct (second panel). Plasmid transcription was labelled by RNA in situ hybridisation using probes to the ß-globin first intron (first panel). Plasmid TDs localised to the interchromatin compartment as shown by the lack of red/green colocalisation in the overlay image (third panel). (B) Punctate plasmid transcription signals (first panel) showed significant overlap with the H2B histone staining (second panel) as shown by the overlay image (third panel). Bars, 5 µm.

View larger version (72K): [in a new window]   Fig. 4. Homologous gene sequences mediate TD formation. (A) HeLa cells were transiently transfected with plasmids pHIV-ß and pHIV- and the plasmid transcription sites were labelled by RNA in situ hybridisation with probes to the ß-globin (first panel) and 2-globin (second panel) first introns. The plasmids formed separate TDs as shown by the absence of red-green colocalisation in the overlay image (third panel). Bar, 1 µm. (B) Line scans showing the relationship between the pHIV-ß (green) and pHIV- (red) transcription signals. The scans are 2D intensity plots corresponding to the yellow lines in Fig. 2A and are labelled as (i) and (ii) accordingly. There is minimal correlation between the pHIV-ß and pHIV- signals and the signal peaks do not overlap. (C) HeLa cells were transiently transfected with plasmids pHIV-ß and pHIV-ß, which share 1243 bp of homologous ß-globin gene sequence, and the transcription sites were labelled by RNA in situ hybridisation with probes to the ß-globin (first panel) and 2-globin (second panel) first introns. The plasmids formed shared TDs as shown by the yellow signals in the overlay image (third panel). Bar, 5 µm. (D) Line scans showing the relationship between the pHIV-ß (green) and pHIV- (red) transcription signals. The scans are 2D intensity plots corresponding to the yellow lines in Fig. 2C and are labelled as (i) and (ii) accordingly. The plots indicate that there is significant correlation between the pHIV-ß and pHIV-ß signals.

View larger version (26K): [in a new window]   Fig. 5. TD formation correlates with the length of homologous ß-globin gene sequence. (A) Plasmid maps showing the design of the ß plasmids. Boxes represent promoters and exons, black arrows indicate transcriptional start sites, and `pA' indicates polyadenylation sites. The plasmids were based on the pUC18 backbone (not shown) and contained the HIV-LTR promoter upstream of the plasmid gene sequences. ß-globin sequences are shown in green while 2-globin sequences are shown in red. (B) Plasmid pHIV-ß was transiently transfected into HeLa cells with each of the ß plasmids, and the TDs were labelled by RNA in situ hybridisation using ß-globin and 2-globin intronic probes. The degree of colocalisation between the TDs was calculated using the overlap coefficient which ranges from 0 to 1, with 1 indicating perfect colocalisation. The blue curve indicates the length of ß-globin gene sequence in the co-transfected plasmids. The highest overlap coefficients were observed when pHIV-ß was transfected alone and the nascent transcripts were double-labelled with two different probes to the ß-globin first intron (bar 1). Co-transfections of pHIV-ß and plasmid pHIV- (which contains no ß-globin gene sequence) gave the lowest overlap coefficients (bar 2). When pHIV-ß was co-transfected with plasmids containing varying lengths of ß-globin gene sequence (bars 3-7) the value of the overlap coefficient correlated with the length of homologous ß-globin gene sequence. (C) Homologous 2-globin gene sequences can also mediate TD formation. Plasmid pHIV- was co-transfected with each of the ß plasmids shown in Fig. 3A. The TDs were labelled by RNA in situ hybridisation using ß-globin and 2-globin intronic probes. The degree of colocalisation between the transcription signals was measured using the overlap coefficient. The blue curve indicates the length of 2-globin sequence in the co-transfected plasmids. The highest overlap coefficients were observed when pHIV- was transfected alone and the nascent transcripts were double-labelled using two different 2-globin first intron probes (bar 1). Co-transfections of pHIV- and plasmid pHIV-ß gave the lowest overlap coefficients (bar 2). When pHIV- was co-transfected with plasmids containing varying lengths of 2-globin gene sequence (bars 3-7) the value of the overlap coefficient correlated with the length of homologous 2-globin gene sequence.

View larger version (54K): [in a new window]   Fig. 6. High plasmid-copy numbers favour punctate plasmid transcription. (A) HeLa cells were transiently transfected with increasing concentrations of plasmid pHIV-ß and the plasmid transcription sites were labelled by RNA in situ hybridisation using probes of the ß-globin first intron. Higher DNA concentrations correlated with higher transfection efficiencies (dashed blue curve) and also with a greater percentage of transfected cells displaying punctate plasmid transcription signals (pink curve). (B) HeLa cells were transiently transfected with pHIV- and pHIV-ß at a concentration ratio of 9:1. The cell in this image displays pHIV-ß in TDs and pHIV- in punctate transcription sites indicating that high plasmid copy numbers do not inhibit TD formation by non-homologous plasmid in the same cell. Bar, 5 µm. (C) Plasmid pCMV-GFP-ß was transiently transfected into HeLa cells and the plasmid transcription sites were labelled by RNA in situ hybridisation using probes to the ß-globin first intron. RNA and GFP-ß-globin signal intensities were recorded for individual cells and plotted according to the plasmid transcription pattern. Results were normalised to the signal intensities observed in cells with TDs. Cells with punctate plasmid transcription showed significantly elevated levels of GFP staining relative to cells with TDs. A cell transfected with pCMV-GFP-ß displaying plasmid TDs (red) and GFP signals (green) is shown on the right. The overlapping GFP signals and TDs appear yellow. Bar, 5 µm.

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