Cameron S Osborne ¹, Lyubomira Chakalova ¹, Karen E Brown ², David Carter ^{1, 4}, Alice Horton ¹, Emmanuel Debrand ¹, Beatriz Goyenechea ¹, Jennifer A Mitchell ¹, Susana Lopes ^{3, 4}, Wolf Reik ³, and Peter Fraser ¹

¹ Laboratory of Chromatin and Gene Expression, The Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, UK.
² Chromosome Biology Group, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 ONN, UK.
³ Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, UK.
⁴ Present addresses: Sir William Dunn School of Pathology, Oxford University, South Parks Road, Oxford, OX1 3RE, UK (D.C.); The Wellcome Trust/Cancer Research UK Institute, Tennis Court Road, Cambridge, CB2 1QR, UK (S.L.).

Correspondence should be addressed to Peter Fraser:   peter.fraser@bbsrc.ac.uk 

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites. 

Figure 1. Transcription frequencies of genes on mouse chromosome 7.

(a) Schematic map of distal region of mouse chromosome 7 showing the positions of the Hbb gene cluster (96.0 Mb from centromere), Eraf (120.4 Mb), Uros (125.6 Mb), Igf2 (134.7 Mb) and Kcnq1ot1 (135.3 Mb). Asterisks indicate the locations of additional primers used in 3C analysis (Fig. 7). (b) RNA FISH on adult anemic spleen erythroid cells with intron probes for Uros. Note cell nuclei with zero, one or two signals (green). DAPI staining is blue. Scale bar, 5 mm. (c) The percentage of alleles with a gene transcription signal by RNA FISH for Hbb-b1, Hba, Eraf, Uros, Igf2 and Kcnq1ot1 in erythroid cells. (d) Relative primary transcript levels for Hbb-b1, Eraf, Uros and Igf2, measured by quantitative RT-PCR of intron sequences. Average values are shown with Eraf set to 1.

Figure 2. Colocalization of transcribed genes on mouse chromosome 7.
Double-label RNA FISH for Hbb-b1 gene transcripts and various other genes. Red signals are Hbb-b1 and green signals are Eraf (a), Uros (b), Igf2 (c), Kcnq1ot1 (d) and Hba (e). The percentage of signals that overlap with an Hbb-b1 signal is given at the bottom of each panel. DAPI staining is blue. Scale bar, 5 mm.

Figure 3. Colocalization of genes is transcription-dependent.
Confocal microscopy and 3D measurement of separation distances between Hbb-b1 and Eraf. (a) RNA FISH 3D reconstructed image from confocal stack on anemic spleen erythroid cell. Hbb-b1 transcription foci (red) and Eraf (green). Scale bar, 5 mm. (b) DNA FISH 3D reconstructed image from confocal stack on anemic spleen erythroid cell. Hbb (red) and Eraf (green). (c) Box and whiskers plot of the distributions of 3D measurements of the separation distance between Hbb-b1 and Eraf using RNA FISH (n = 79) and DNA FISH (n = 130). Lower and upper whiskers denote the 10th and 90th percentiles, respectively, of the distribution. The lower and upper limits of the boxes denote the 25th and 75th percentiles, respectively. Solid and dashed lines in the boxes indicate the median and mean, respectively. Outliers (i.e., values above and below the 10th and 90th percentiles) are shown as filled circles.

Figure 4. Actively transcribed genes associate with RNAP II foci.
(a) RNA immuno-FISH of Hbb-b1 transcription (red) with RNAP II staining (green) in anemic spleen erythroid cells. Scale bar, 5 mm. (b) DNA immuno-FISH of Eraf (red) with RNAP II staining (green). (c) Comparison of the percentage of alleles exhibiting a gene transcription signal by RNA FISH (black), with the percentage of loci that overlap with an RNAP II focus by DNA FISH (gray) for Hbb-b1 (n = 83), Eraf (n = 59), Uros (n = 47) and P2ry6 (n = 79).

Figure 5. Actively transcribed genes colocalize to shared transcription factories.
(a) Single optical section of a triple-label DNA immuno-FISH on erythroid cell, showing Hbb (green), Eraf (red) and RNAP II foci (blue). The merged and separate channels of the signals are shown in the side panels. On the left of the main panel, an Hbb signal alone associates with an RNAP II focus. On the right, two colocalizing signals associate with the same RNAP II focus. Scale bar, 5 mm. (b) A separate optical section of the same cell showing the second Eraf allele, which does not associate with an RNAP II focus. (c) Box and whiskers plot of the distributions of 3D measurements of the separation distance between Hbb and Eraf loci (n = 84), divided into RNAP II–associated versus nonassociated. (d) Triple-label RNA immuno-FISH on erythroid cell showing Hbb-b1 (red), Eraf (green) and RNAP II (blue). Left panels, colocalized transcription signals associating with the same RNAP II focus. Right panels, separate transcription signals associating with distant RNAP II foci.

Figure 6. Comparison of RNAP II foci in several tissue types and MEFs.
(a) Deconvoluted maximum-intensity projections of image stacks of nuclei immunostained for RNAP II. E10, embryonic blood; E14, fetal liver erythroid; AS, adult anemic spleen erythroid; Sp, normal adult spleen; Th, adult thymus; Br, fetal brain. Scale bar, 10 mm. (b) Numbers of RNAP II foci counted for each nucleus shown in a.

Figure 7. 3C analysis.
PCR detection of unique ligation products between Hbb-b1 and various restriction fragments in E18.5 fetal liver (E) and fetal brain cells (B). The nuclei were fixed for either 5 min (a) or 10 min (b). The restriction fragments assayed were the Hbb LCR, three intervening genomic regions (104, 116, 124; shown in Fig. 1) and Eraf, Uros and Igf2. Detection of ligated products between two restriction fragments of Calr was used as a positive control for fetal liver and brain, as described [29]. M, DNA size marker.

Figure 8. Model of dynamic associations of genes with transcription factories.

Schematic representation of chromatin loops (black) extruding from a chromosome territory (gray). Transcribed genes (white) in RNAP II factories (black circles). Potentiated genes (free loops) that are not associated with RNAP II factories are temporarily not transcribed. Potentiated genes can migrate to a limited number of preassembled RNAP II factories to be transcribed (dotted arrows). We propose that both cis and trans associations are possible.

http://www.nature.com/ng/journal/v36/n10/suppinfo/ng1423_S1.html

1. Zink D, Amaral MD, Englmann A, Lang S, Clarke LA, Rudolph C, Alt F, Luther K, Braz C, Sadoni N, Rosenecker J, and Schindelhauer D, "Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei".

2. Gilbert N, Boyle S, Fiegler H, Woodfine K, Carter NP, and Bickmore WA, "Chromatin Architecture of the Human Genome: Gene-Rich Domains Are Enriched in Open Chromatin Fibers".

3. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

4. Parada LA, McQueen PG,  and Misteli T, "Tissue-specific spatial organization of genomes".

5. Chambeyron S,  and Bickmore WA, "Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription".

6. Kreth G, Finsterle J, von Hase J, Cremer M, and Cremer C, "Radial Arrangement of Chromosome Territories in Human Cell Nuclei: A Computer Model Approach Based on Gene Density Indicates a Probabilistic Global Positioning Code".

7. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming".

8. Hovsepian JA, and Frenster JH, "Euchromatin as an Extensile Force within Mammalian Cell Nuclei".

9. Hovsepian JA, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".
 

Links to RNA and Biological Causality:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".