rescue of mutant phenotypes when testing for intragenic complementation. This allows for an assessment of trans- vection at essentially the single molecule level, because each transgene pair is present as a single copy in each cell. Although the system we have described here should be very valuable for studying transvection, there is one signiﬁcant drawback. First, using the phiC31 integrase system along with engineered transgenes requires that all tested sequences be removed from their endogenous locus and combined with other foreign sequence elements. Thus, CRMs may behave very differently in this system than they do in their endoge- nous locus. It is therefore encouraging that changing the promoters in our transgenes did not eliminate transvection effects, and the patterns of transvection were qualitatively similar between two distant attP sites for the pairs that were tested at both sites (Figure 2, D and E; D. J. Mellert, unpublished observations). These observations suggest that transvection is robust to the surrounding genomic environment. A second limitation of our study, but not the transgenic system we propose per se, is that it did not include the use of chromosomal rearrangements that disrupt pairing, which is usually the test for the pairing dependence of transvection effects. This raises the question as to whether the interac- tions we see are actually pairing dependent. That both trans- genes require insertion into the same genomic location is strong evidence for this possibility. The prevailing view of transvection is that proximity of the two participating alleles is required. Although it is formally possible that two trans- genes that substantially differ in sequence could interact even in the presence of chromosomal rearrangements (for an example of such a strong transvection effect, see Hopmann et al. 1995), it is not clear how transvection would be position dependent in such a case, because it would imply both trans- genes would have the ability to associate independent of ﬂanking homology. It has been shown that it is possible for two alleles to participate in transvection across large chromo- somal distances or between chromosomes, but only if there is sufﬁcient contiguous ﬂanking homology (Ou et al. 2009). These ambiguities could be resolved in future studies. The phiC31 system for studying transvection is amenable to more classic techniques using chromosomal rearrange- ments, as it would allow for many different transgenes to be inserted into the same location on a rearranged chromo- some. This could be used to test differences between CRMs in the robustness of transvection to various chromosomal rearrangements. Despite the preceding caveats, we have provided some insight into the mechanism of transvection using the phiC31- integration strategy. One particularly surprising feature of transvection is that it can occur reciprocally between two fully functional alleles. This has been a difﬁcult question to address because it requires the ability to assess transcription of each allele independently, and most studies of transvection depend on intragenic complementation between one allele with a disrupted regulatory region and one allele with a dis- 1138 D. J. Mellert and J. W. Truman rupted coding sequence (however, see Goldsborough and Kornberg 1996). We have shown that enhancers can act in trans, even if they are in cis to a fully functioning and strong core promoter and an intact coding sequence. This is consistent with previous reports that have shown that enhancers may work on trans promoters in the presence of a functional cis promoter (Goldsborough and Kornberg 1996; Casares et al. 1997; Sipos et al. 1998). Thus en- hancer preference for the cis promoter does not necessarily prevent transvection between wild-type alleles, supporting the possibility that transvection is common throughout the wild-type Drosophila genome. Further supporting this view, we found that 50% of CRMs we tested could un- dergo transvection when paired with at least one of three different transgenes. Given that most genes contain mul- tiple CRMs in their regulatory regions and that the genome is generally permissible for transvection, it is probably the case that most genes contain CRMs that are competent to undergo some degree of transvection. Our ability to observe the activity of enhancers at single cell resolution also reveals something about the dynamics of enhancer action. In cases where both promoter strengths are equal, some CRMs drive expression of both GAL4 and LexA in the same cell. This means that in some circumstances, a single enhancer can activate both paired promoters. Thus, when transvection is directional due to differences in pro- moter strength, it is not the case that the stronger promoter locks its cis enhancer into a conﬁguration that physically prevents it from interacting with the weaker trans promoter. Rather, the stronger promoter probably outcompetes the weaker promoter for either enhancer input or transcription initiation factors that are present at a limiting local concen- tration (Morris et al. 1999). On the other hand, our data show that an enhancer can still act on its own weak cis promoter even in the presence of a strong trans promoter, demonstrating that enhancers exhibit some degree of cis preference even when there is competition between paired promoters. The ﬁnding that UAS sequences can drive transcription in trans in the presence of GAL4 protein also shows that trans- vection does not necessarily require sequence homology be- tween genes/transgenes, because somatic pairing and ﬂanking homology will still ensure that they are in close proximity in the nucleus. Moreover, it is possible that there is no sequence that uniquely confers the ability of an en- hancer to act in trans. The ability to bind a transcriptional activator is sufﬁcient. This suggests to us a model for trans- vection in which any transcriptional activator can act in trans provided (1) the trans promoter is competent to initi- ate transcription in a given cell type (i.e., not epigenetically silenced, can recruit and bind RNA polymerase II, etc.), and (2) there is not active repression by proteins bound to the trans regulatory region. We stress some caution with this interpretation, however. All of our transgenes were inserted into the genome along with copies of mini-white and yellow (Figure 1A). Because pairing-dependent trans effects have