A biophysical model for transcription factories
© Canals-Hamann et al.; licensee BioMed Central Ltd. 2013
Received: 14 August 2012
Accepted: 5 December 2012
Published: 9 February 2013
Transcription factories are nuclear domains where gene transcription takes placealthough the molecular basis for their formation and maintenance are unknown. In thisstudy, we explored how the properties of chromatin as a polymer may contribute to thestructure of transcription factories. We found that transcriptional active chromatincontains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Singlefibre analysis showed that this modification spans the entire body of the gene.Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active andinactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in thechromatin fibre. The result of this change in flexibility is that chromatin couldbehave like a multi-block copolymer with repetitions of stiff-flexible(active-inactive chromatin) components. Copolymers with such structure self-organizethrough spontaneous phase separation into microdomains. Consistent with such modelH4K16ac chromatin form foci that associates with nascent transcripts. We propose thattranscription factories are the result of the spontaneous concentration of H4K16acchromatin that are in proximity, mainly in cis.
KeywordsEpigenetics Biophysics H4K16Ac BrUTP Transcription Factories RNA pol II Nuclear organization
Transcription in eukaryotes is organized in transcription factories (TFs), which arenuclear domains where several genes are grouped to be transcribed together [1, 2][3–5]. The current opinion is that the genes in a TF interact by a loopingmechanism [5–8]. It has been suggested that chromatin looping plays an important role incontrolling gene activity by bringing together promoters and enhancers or TFs . Some studies suggest that promoter-enhancer loops are maintained by theinteraction of proteins associated with these cis-regulatory elements . This interaction precedes chromatin activation, which is required for generelocation to the TF by an unknown mechanism . It has been proposed that TFs are maintained by depletion attraction forces(excluding volume effect) between RNA pol II molecules . However, experimental evidence has shown that genes remain at the factoryeven when active RNA pol II is not present . This makes the excluding volume model very improbable and suggests that thisstructure is not the result of transcription. Instead experimental evidence points tohistone acetylation as being responsible for loop formation . For these reasons we explored the possible contribution of chromatinacetylation in the formation of TFs.
Chromatin at the TF is decondensed  and contains active transcription marks like histone acetylation or H3K36me3 . Among all the possible Lysine residues that can be acetylated, H4K16Ac isvery special because it prevents the formation of compacted chromatin by inhibiting theinter-fibre interaction [15–18]. Moreover, H4K16 acetylation is associated with both active chromatin  and with the active transcription marker H3K4me3 [19–21].
Results and discussion
The chromatin spreading technique allowed us to measure the length of H4K16Ac tracks.The distribution of H4K16Ac stretches showed a lognormal distribution with average sizeof ~15 Kb (Figure 1e). H4K16Ac tracks rarely appeared isolated,instead they tended to cluster, spanning several hundreds of Kb (348 ± 90; range235–530 Kb) (Figure 1d). The extension of the gaps betweentwo consecutive H4K16Ac tracks in the cluster showed a lognormal distribution with anaverage distance of ~30 Kb (Figure 1f). The analysis of thepolymerases loaded onto H4K16Ac tracks showed that not all the tracks were stained withBr-RNA or P-RNA pol II. The number of nascent transcripts or P-RNA pol II per track waslow (0.7 ± 1 transcripts/track and 0.8 ± 0.9 P-RNA pol II/track). This was inaccordance with our previous findings, suggesting that most of the TUs contain onemolecule of RNA pol II . The fact that some H4K16Ac tracks of chromatin were not associated to RNApol II or Br-RNA could reflect a temporal discrepancy between the transcription andacetylation processes of chromatin. Indeed, transcription by RNA pol II takes only a fewminutes [25–27] while deacetylation of active chromatin can take several hours , providing a molecular memory of recently-transcribed chromatin. On the otherhand, H4K16Ac tracks are not a special feature of lymphocytes as we were able to findthe same chromatin organisation in all the mammalian cell types tested including: Hela,Epstein Barr transformed lymphocytes, human lymphocytes, primary human fibroblasts,primary mouse fibroblasts and murine erythroleukemia cells (both differentiated andundifferentiated).
The clusters in all the different cell types analysed were identical with respect to thenumber of TUs (8 ± 2 TUs/Cluster), suggesting that co-linear active genes expressedat the same time, in agreement with the analysis of expression data using FDCP mix cells . The sliding window analysis (applying a window of 500 Kb) over the entiregenome showed that genes are active in clusters (Figure 1g), inaccordance with our chromatin spreads data. Moreover, our results are consistent withthe co-expression data after a Serial Analysis of Gene Expression where the cluster sizewas <500 Kb . From these data we can conclude that co-linear TUs are active at the sametime in the same cell.
How are these TUs organised in the cell nucleus?
A prediction of the multi-block copolymer model is that microphase separation mustpersist as long as H4K16Ac is present in chromatin. In fact, H4K16Ac foci wereunperturbed by treatments like 2 M NaCl extraction, which disrupts chromatin;transcription inhibition by DRB (5,6-Dichlorobenzimidazole1-β-D-ribofuranoside), which reduces RNA pol II transcription by 98%; and heatshock (1h 45°C), which releases RNA pol II from the DNA  (Additional file 1: Figure S1). The only way todisrupt these foci was by formamide treatment, which works as a solvent for theelectrostatic self-assembled polymers (Additional file 2:Figure S2).
These experiments contradict the excluding volume model  and are in agreement with the multi-block microphase separation hypothesisproposed in this study.
The next question about the genes in a TF is where they come from. Several studieshave shown that genes in cis and in trans are able to interact in the same TF [5–7]. However, the analysis of chromatin spreads showed that collinear genesare active in the same cell at the same time. This guarantees that several H4K16Actracks are in close proximity. Therefore, most of these collinear TUs would probablyaggregate in the same microsphere, as occurs in similar situations with themulti-block copolymers . The experimental evidence from chromosome configuration capture analysissuggests that local chromatin is the primary source of interaction for any genomicloci . Nevertheless, we cannot exclude the possibility that some genes locatedfurther away in the same chromosome or in another chromosome can interact due toproximity or chromatin folding.
Finally, a remarkable feature of TFs is their constant size across species anddifferentiation stages . According to the multi-block copolymer model for chromatin organisation,the way to change the size of H4K16Ac foci (and consequently TFs) is by increasingthe number of active genes in a given region or by unrestricting the mobility of theactive chromatin. The latter has been reported in experiments using plasmids thatrendered larger TFs than the endogenous ones [35, 36].
In summary, we present evidence of the relationship between epigenetic marks and theTF structure. Our model proposes that active chromatin self-organises in the nucleusdue to the special physical properties of H4K16Ac modified chromatin. Therefore, ourmodel implies that chromatin becomes activated (H4K16Ac modified) before joining aTF. This is conceptually very different from current transcription factory model,which proposes that genes are targeted to TFs to “enhance production byconcentrating the relevant machines, resources, and expertise in one place” .
Materials and methods
Transcription in vivo and in vitro
For in vivo transcription, cells grown on coverslips were incubated in presence of2.5 mM BrU for several min.
For in vitro transcription, cells grown on coverslips were treated as described .
Cells (103 cells in 5 μl) were spotted onto a 22 × 50mm glass slide and 5 μl of lyses buffer were added (Lyses buffer: 1%sarkosyl, 25 U/ml ribonuclease inhibitor, 10 mM EDTA, and 100 mM Tris–HCl (pH7.4)). After 10 min at 20°C, the slide was tilted to allow the drop to run down.Samples were air-dried and fixed in 4% Paraformaldehyde for 10 min. Clusters weredefined as two o more hyper acetylated tracks in less than 100 Kb. For quantificationof clusters of hyper acetylated chromatin between 150 and 200 tracks of hyperacetylated chromatin were analysed.
After blocking for non-specific antibody binding, immunolabelling was carried out asdescribed . For detection of primary transcripts, we used mouse anti-IdU/BrdU (5mg/ml; Caltag Laboratories, Burlingame, CA). For detection of H4K16ac we usedantibodies raised in rabbit and mouse (Serotec, Kidlington, UK, Abcam). RNA pol IIhyperphosphorylated in Ser 2 was detected with H5 antibody (Covance). Secondaryantibodies donkey anti-mouse IgG or IgM tagged with Cy3 (1/200 dilution; JacksonImmunoResearch, Bar Harbor, ME) and donkey anti-rabbit IgG tagged with Alexa 488(1/200; prepared using a Molecular Probes kit, Inc., Eugene, OR). DNA staining wasperformed with 200 nM TOPRO-3 (Molecular Probes) for 5 min. Then coverslips weremounted on slides using Vectashield (Vector laboratories), and images were collectedusing a Radiance 2000 confocal microscope (Bio-Rad Laboratories, Hemel Hempstead,Herts, UK), Distances were measured using EasiVision software (Soft Imaging SystemsGmbH, Münster, Germany) and data exported to Excel (Microsoft) for analysis.
The degree of spreading of the chromatin was measured by hybridising the spreads witha fragment of DNA of 47.26 Kb; the spreading was 3.9 + 0.2 Kb/μm.
Microarrays and sliding window analysis
Mouse FCDP mix cells were used. cRNA synthesis and hybridisation to oligonucleotidearray were performed as described .
The sliding window analysis was performed by applying a window of 500 Kb over thechromosomes and moved at 5 Kb steps along a chromosome to know whether the genescontained in that window were more likely to be transcribed together than just bychance.
The authors would like to thank Elspeth McFarlane for helping with the English. To:Ministerio de Economia y Competitividad, (Spain) (Grant number: BFU2009-10792) andThe Medical Research Council (UK), for supporting this work. We also thankFundação Ciência e Tecnologia, Portugal for funding RPN.
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