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 TH
1 and TH
2 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 TH
2 locus caused a delay, rather than a reduction, in the expression of the TH
1 gene, and intriguingly this effect was measurable only in differentiating TH
1 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
) 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
expression, formal evidence for interchromosomal enhancer activity awaits demonstration that the chromosomal deletion of one of these repeats causes a drop in IFN
expression. Finally, the imprinting control region (ICR) of the H19
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; ). 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.