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Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra β-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous β-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in ‘jackpot’ cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression.
High-resolution profiling of transcription-factor binding sites, the discovery of conserved genetic elements and identification of regulatory sites using technologies such as DNaseI hypersensitive site mapping1, has demonstrated that the number of genomic sites with transcription regulatory potential far exceeds the number of genes. One of the main challenges of the post-genomic era therefore is to assign function to each element. This requires understanding of the capacity of regulatory sites to reach over distance and identify specific target genes at the single-cell level. It is known that mammalian DNA elements can modulate the activity of distant genes on the same chromosome, up to a genomic distance of over a megabase2,3. The three-dimensional structure of the mammalian genome facilitates long-range gene regulation. This was first shown for the β-globin locus, which contains multiple β-globin genes arranged in the order of their developmental expression (Fig. 1a). Upstream of the genes resides a large regulatory DNA element that enhances expression of the β-globin genes up to ~100-fold4. The element is called a locus control region (LCR), as it has the capacity to confer position-independent and copy-number-dependent expression on reporter genes in transgenic assays in addition to its classical enhancer activity5. Otherwise, the β-globin LCR is not different from classical enhancers: it upregulates gene expression over distance, it functions in a tissue-specific manner and genes compete for its activity6,7. At its endogenous location, the LCR enhances expression of the β-globin genes through physical interactions, thereby looping out the intervening DNA that may carry non-responding genes8–10 (Supplementary Fig. S1). A similar mode of action involving chromatin looping has been shown for enhancers in several other gene loci11,12.
At a higher-order level of organization, microscopy studies have shown that genes can occupy differential positions in the nucleus depending on their expression status13–15 and that regulatory DNA elements are instrumental in targeting these genomic regions to specific nuclear positions16–19. Moreover, recent observations suggest that functionally related genes on the same and different chromosomes may come together in the nuclear space for co-transcription20. Finally, some reports have suggested functional communication between regulatory sites located on different chromosomes21–25. Collectively, these studies raise questions on how genes and regulatory sequences explore the nuclear space to engage in functional crosstalk with preferred genomic partners.
To investigate the ability of regulatory DNA elements to relocate chromosomal regions in the nuclear interior and search for preferred target genes, we used transgenic mice with the human erythroid-specific β-globin LCR (hLCR) site-specifically integrated into a gene-dense region on chromosome 8. This region, 8C3–C4, contains many housekeeping genes18. Two transgenic lines were available, LCR-S and LCR-AS, each carrying the hLCR without globin genes at 8C3–C4, but in opposite orientations (Fig. 1b). In a previous study we showed that in erythroid cells each LCR detectably upregulates several housekeeping genes that directly surround the integration site up to sixfold, with the most distal one being 150 kilobases (kb) away. Microscopy studies revealed that both LCRs positioned 8C3–C4 more often outside its own chromosome territory, raising the question of to where the LCR migrates its integration site.
We reasoned that, in the most extreme situation, the hLCR could ‘search’ for a natural target gene present anywhere in the genome, including on its homologous chromosome, as is seen in transvection. The term transvection was coined to describe transcriptional regulation across (paired) homologous chromosomes, a phenomenon mainly studied in Drosophila, where homologues frequently pair26,27. Pairing is generally not observed in mammals28, but may occur in special instances where mono-allelic expression needs to be ensured29–31. As mammalian chromosomes intermingle extensively32, individual chromosomal segments may indeed invade the territories of other, possibly homologous, chromosomes. We generated transgenic mice carrying a human Aγ-globin reporter gene in one or the other orientation at exactly the same location in 8C3–C4 (Fig. 1b). The Aγ-globin gene is a human fetal globin gene that, in mouse transgenics carrying a full human globin cluster including the hLCR, is most highly expressed between embryonic day 10.5 and 12.5 (ref. 33; Fig. 1a). Crossing the Aγ-globin reporter mice with transgenic mice carrying the hLCR on the homologue revealed no increase in Aγ-globin gene expression at any of the relevant developmental stages (Fig. 1c). Fluorescence in situ hybridization (FISH) experiments also did not reveal increased interaction between the homologues (Fig. 1d,e). Therefore, our experimental system provides no evidence for mammalian transvection involving the hLCR at 8C3–C4. Although these data do not rule out mammalian transvection at other genomic locations, they do strongly suggest that the ectopic hLCR does not have unlimited freedom to search the nuclear interior for natural target genes.
To investigate in more detail the ability of the hLCR to actively determine its genomic environment, we applied chromosome conformation capture34 on chip (4C) technology to E14.5 fetal livers of wild-type and homozygous LCR-AS mice (Supplementary Fig. S2). 4C captures and identifies spatially proximal DNA sequences to enable an unbiased scan for DNA elements interacting with a locus of choice35. Analysis of the 4C data revealed that the ratio of inter-over intrachromosomal captures increased in the LCR-AS mice (Supplementary Fig. S3), in agreement with the hLCR causing looping out from the chromosome territory (ref. 18). This might be a reflection of the LCR actively engaging in interactions with new interchromosomal partners. To identify specific DNA interactions, sliding-window algorithms were applied that scan the linear chromosome templates for significant clustering of independently captured sequences36,37. Using both conventional analysis and a newly developed high-resolution analysis, we identified a highly similar set of interacting regions for 8C3–C4 with or without an integrated hLCR, both on the same chromosome (in cis) and on different chromosomes (in trans) (Fig. 2a–c and Supplementary Figs S4, S5a). Extensive fluorescence in situ hybridization experiments on cryo-sections (cryo-FISH; ref. 32) validated the 4C data (Fig. 2d,e). Some regions in trans showed increased interaction frequencies with the hLCR, again in agreement with the LCR causing looping out of the chromosome territory. However, we did not find chromosomal regions that exclusively interacted with the hLCR. Altogether, this demonstrates that the hLCR at 8C3–C4 does not search the genome to contact new preferred interaction partners. Rather, the chromosomal context of 8C3–C4 dictates the nuclear space that can be explored by the integrated hLCR.
Whereas the overall genomic environment did not change, a quantitative comparison of 4C results showed that the hLCR at 8C3–C4 captured a subset of pre-existing interchromosomal interaction partners more efficiently. This was most obvious for the α-globin locus on chromosome 11, but also for the endogenous β-globin locus on chromosome 7 (Fig. 3a). Homology between the human and mouse LCRs is limited (<10%) and therefore not expected to underlie the latter contact. The two loci have in common that they are very highly expressed and carry genes controlled by EKLF and GATA-1, two transcription factors that also bind to the LCR. Ranking genomic regions on the basis of their difference in 4C signal revealed significant enrichment (P < 0.01, hypergeometric test) of EKLF (refs 38,39)- and GATA-1 (ref. 40)-regulated genes, as well as of highly expressed genes, among regions contacted more strongly by the LCR (Supplementary Table S1). The same conclusion was drawn from an analysis per microarray probe (Fig. 3b). Each category of genes seems to independently attract the LCR (Fig. 3c and Supplementary Fig. S5b). We conclude that the LCR at 8C3–C4 behaves like a ‘dog on a lead’: the chromosomal context of 8C3–C4 imposes important constraints on its freedom to move, but within the restricted nuclear subcompartment that it occupies the LCR preferentially contacts genes that are controlled by shared transcription factors.
Notwithstanding its restricted ability to change the spatial environment of 8C3–C4, the ectopic LCR is involved in many long-range interactions in cis and trans. We investigated whether any of the contacted genes were upregulated by the LCR. For this, we profiled the transcriptomes of wild-type and LCR-transgenic littermates using microarrays. A small number of genes was found to be upregulated more than twofold in transgenics. These were the previously characterized genes proximal to the LCR in cis (ref. 18), plus a single gene in trans, Hbb-bh1, that was confirmed to be upregulated by quantitative PCR with reverse transcription (RT–qPCR) (Fig. 4 and Supplementary Fig. S6a–d).
Interestingly, Hbb-bh1, or βh1, is one of the endogenous β-globin genes normally expressed at high, LCR-dependent levels earlier in development, in embryonic blood cells (Fig. 1a). In E14.5 fetal livers the gene is looped away from the endogenous LCR (Supplementary Fig. S1). However, three independent probe-sets on the Affymetrix microarray and two independent RT–qPCR primer-sets reveal that the gene is not fully silent, but expressed at basal levels in wild-type fetal livers (Fig. 4 and Supplementary Fig. S6). Importantly, increased Hbb-bh1 expression was consistently found in transgenics carrying the LCR in either one of the orientations when compared with their wild-type littermates (Fig. 4a,b), but not when 8C3–C4 exclusively carried a Neo selection cassette (Fig. 4c). No other β- or α-globin gene detectably changed in expression in the presence of the ectopic LCR (but see below), as judged by microarray analysis and RT–qPCR strategies that measure gene expression across cell populations (Fig. 4d and Supplementary Fig. S6e).
Given that the endogenous β-globin locus carrying βh1 is among the chromosomal regions that are preferentially contacted by the ectopic LCR (Fig. 3a), we hypothesized that such interchromosomal LCR–βh1 contacts drive increased βh1 expression. DNA and RNA-FISH analysis showed that the interaction occurs in 5–10% of the cells, whereas higher-resolution cryo-FISH identified contacts between 2 and 3% of the alleles (Supplementary Table S2). Irrespectively, this raises the question of how these relatively rare interchromosomal contacts can account for the overall twofold increase in transcript levels measured across the entire cell population. We reasoned that interaction frequencies measured by FISH in fixed cells may reflect chromatin dynamics, such that over time a given interaction occurs in every cell. Alternatively, they reflect genome conformations that are cell specific and relatively stable after mitotic exit, implying that, in a given nucleus, genomic loci sample overlapping or non-overlapping nuclear subvolumes. If the latter were true, ‘jackpot’ cells should exist with accumulated βh1 messenger RNA levels in combination with frequent interchromosomal LCR–βh1 interactions. To investigate this we carried out RNA FISH using a mixture of probes visualizing both primary transcripts and accumulated mRNA (ref. 41; Fig. 5a,b). In wild-type E14.5 fetal liver cells, we failed to detect the βh1 primary transcript signals that were visible in E10.5 embryonic blood cells (Supplementary Fig. S7), consistent with its marked drop in expression during development. Accumulated cytoplasmic βh1 mRNA was seen in only a small percentage (3.5%) of cells. These cells also contained the highest adult β-globin transcript levels (Hbb-b1, or β-major) and we therefore assumed they represent the most differentiated erythroid cells in the liver that had most time to accumulate β-globin mRNA. Notably, in only one out of 50 of these cells was an interchromosomal interaction between 8C3–C4 and β-globin observed, which does not exceed the interaction frequency of 5.1% measured across the entire wild-type red-blood-cell population (Fig. 5c and Supplementary Table S2). On the other hand, in LCR transgenic fetal liver cells the percentage of cells with high βh1 mRNA levels is increased to 7.0%. More importantly, in these transgenics 15/50 cells (30%) with high βh1 levels showed an interchromosomal interaction between 8C3–C4 (with the LCR) and β-globin, a highly significant (P = 6.6 × 10−7, hypergeometric test) increase in interaction frequency when compared with the 8.5% measured across all red blood cells in the transgenics (Fig. 5c and Supplementary Fig. S8a,b). As a control, we analysed α-versus β-globin co-localization in transgenic cells and found no correlation between this interchromosomal interaction and βh1 expression levels (Supplementary Fig. S8c).
We next reversed the question and asked whether cells showing the interchromosomal LCR–βh1 interaction also had increased βh1 transcript levels in their cytoplasm as compared with other red blood cells in the transgenic fetal livers. For this, we developed automated image-analysis software (see Methods) and analysed thousands of cells with respect to their intensity of βh1 RNA FISH signal in the cytoplasm. The analysis showed that in transgenics, but not in wild type, cells with the interchromosomal interaction between 8C3–C4 and the β-globin locus more often have high βh1 levels than the other cells from the same tissue (Fig. 6a). Interestingly, albeit less pronounced, the same was found for β-major (Fig. 6b), the adult β-globin gene located next to βh1 in the endogenous globin locus (Fig. 1a). Its natural extremely high expression level precluded uncovering an extra contribution from the few cells with this interchromosomal interaction by cell-population-based expression assays such as microarrays or RT–qPCR. However, our analysis at the single-cell level revealed that also this adult β-globin gene benefits from contacts with the extra copy of the LCR. βh1 is looped away from the endogenous mouse LCR and as such may be available for contacts with the ectopic human LCR. β-major dynamically forms and breaks contacts with the endogenous LCR (ref. 7), possibly providing opportunity for the ectopic LCR to also engage in contacts and further boost transcription. The genes encoding α-globin did not benefit from contacts with the ectopic LCR, as judged from the single-cell analysis strategy (Fig. 6c). We conclude that our transgenic mice contain a unique population of cells that have increased levels of mRNA for β-globin because these genes on chromosome 7 are contacted and trans activated by the ectopic LCR on chromosome 8.
One of the main challenges of the post-genomic era is to assign function to genomic sites, many of which have regulatory potential. Clearly, this cannot be done without considering the dynamics and spatial configuration of the genome. Here, we uncover properties of nuclear organization that dictate the action of regulatory elements in nuclear space. Our findings should contribute to a working model of genome function. The results demonstrate that regulatory DNA elements can search for preferred interaction partners, which in the case of the LCR are genes controlled by shared transcription factors. The ability to roam the nucleus is however heavily constrained by the chromosomal context. We predict the same to be true for almost all genomic locations, although the degree of constraint may vary. The concept of chromosomal context heavily influencing a gene’s specific nuclear location seems to contradict more deterministic models of nuclear organization, where functionally related genes are proposed to meet at dedicated nuclear sites20. We cannot exclude that the LCR would have a more notable effect when placed at other genomic locations, or that other enhancers exist that are better capable of repositioning chromosomes and forming specific interchromosomal interactions. We note however that very few, if any, regulatory elements have been described with such a strong influence on gene expression and chromatin organization as the β-globin LCR.
An important finding of this study was that the ectopic, orphan, LCR on chromosome 8 contacted many different genes in cis and in trans, including those sharing a similar set of regulatory proteins, but that no measurable effect on the expression of most of them was detected. This suggests that, in mammals, ultralong-range gene regulation within and between chromosomes will be rare, or at least difficult to measure in cell populations. Two endogenous β-globin genes on chromosome 7 were the exception, as they were both upregulated by the ectopic LCR in cells with the relevant interchromosomal interaction. As these are natural (mouse) target genes of the (human) LCR, promoter compatibility and spatial proximity seem essential for transcription regulation over distance. Interestingly, a few examples exist of endogenous tissue-specific enhancers activating not only target genes but also non-target genes that happen to be in physical proximity to the enhancer42,43. Our results open the possibility that such bystander activation may be more common in the genome, but appreciable only in individual cells that have their genome folded such that an enhancer and gene happen to be within contacting distance.
Our data provide genetic evidence for classical enhancer activity between mammalian chromosomes, where the genetic addition or deletion of a regulatory DNA element on one chromosome causes increased or reduced expression of a physically interacting gene on another chromosome. As such, we provide formal in vivo evidence that mammalian regulatory sites do not need an intervening chromatin fibre to propagate activating signals to responding gene promoters elsewhere in the genome, but that spatial proximity, in combination with enhancer–promoter compatibility, is sufficient for gene activation. Interchromosomal interactions between mammalian regulatory sites and genes have been observed before, but genetic evidence for trans activation was lacking so far. For example, the alternatively expressed TH1 and TH2 cytokine loci were seen to come together before their activation in naive T cells. On differentiation to T-helper 1 or 2 cells, the interactions between these signature loci were lost and the respective genes turned on25. The functional consequences of this interaction seemed complex, however, and different from classical enhancer activity. The deletion of a regulatory element in the TH2 locus caused a delay, rather than a reduction, in the expression of the TH1 gene, and intriguingly this effect was measurable only in differentiating TH1 cells that no longer showed the interchromosomal interaction. The interaction was proposed to prepare loci for proper expression during subsequent T-helper cell specification, an activity not previously assigned to regulatory sites25. Interchromosomal gene regulation by a single enhancer was suggested to control the expression of all ~1,200 olfactory receptor genes spread across the genome23, but deletion of the enhancer demonstrated that the enhancer only affects genes in cis44,45. In another study the activation of human interferon beta (IFN-β) expression in response to viral infection was reported to coincide with interchromosomal interactions with three Alu repeat elements harbouring cryptic NF-κB sites21. Although transfection experiments with plasmids carrying these elements supported the idea that the DNA interactions boost IFN-β expression, formal evidence for interchromosomal enhancer activity awaits demonstration that the chromosomal deletion of one of these repeats causes a drop in IFN-β expression. Finally, the imprinting control region (ICR) of the H19-Igf2 locus has been the subject of several studies on interchromosomal DNA interactions22,24,46. The data did not reveal trans activation and were not necessarily consistent, as each study identified different interchromosomal interactions with different functional outcomes, possibly owing to the use of different cell types and/or experimental approaches.
An interesting observation from our artificial system is that interchromosomal interactions can lead to variegated levels of accumulated transcripts in the individual cells. We propose to term the observed phenomenon that cell-specific long-range DNA interactions cause variable gene expression levels among otherwise identical cells ‘spatial effect variegation’ (SEV; Fig. 6d). Stochastic cell-to-cell variation in gene expression, or transcriptional noise, is common in cell populations47. Our data open the possibility that SEV may be one of the underlying mechanisms of transcriptional noise. In such a scenario, the nature of the interacting region will determine whether gene expression goes up or down in the corresponding cell. This is different from position effect variegation48, where variable expression of ectopically placed genes is classically thought to be caused by repressive effects from directly surrounding chromatin. Future research should indicate if SEV is acting on endogenous genes. If so, it may provide specific cells within a larger population with a mechanism to make autonomous cell-fate decisions, without the need for external signalling.
Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturecellbiology
We thank J. Marteijn and W. Vermeulen for providing Rad23a knockout material, Y. Oz for counting FISH slides, V. Buckle for providing BAC probes, J. van Rheenen and the Hubrecht Imaging Center for help with image analysis and E. Splinter and other members of the group for assistance. This work was financially supported by grants from the Dutch Scientific Organization (NWO) (91204082 and 935170621) and a European Research Council Starting Grant (209700, ‘4C’) to W.d.L.
Note: Supplementary Information is available on the Nature Cell Biology website
AUTHOR CONTRIBUTIONSD.N. and W.d.L. designed the experiments, analysed the data and, with help from E.d.W., wrote the manuscript. D.N. and P.K. carried out experiments. E.d.W. analysed 4C data and developed the automated FISH image analysis. H.v.d.W. analysed 4C and microarray expression data. M.S. carried out 4C experiments. M.L-J. and R.H.S. designed and synthesized RNA-FISH probes. B.E. and A.d.K. helped with the FISH experiments.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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