R24B02 R23G07 R58G08 R54E05 R11G08 R36B03 R54G12 R64F08 R29B04 R16H05 R64E01 R19D12 R33G12 R49D02 R23H08 R26B05 R32G10 R41G09 R44E04 R66B07 R29D02 NAa )c )[ )[ ) [ [ ) ) — — — — — — — — — — — — [b )[ ) Rd [ [ — — [ — — — — — — — — — — — a Crosses between a GAL4 and LexA using the same CRM are not scorable for transvection because they express in the same pattern. b Up arrow indicates LexA is ectopically expressed in elements of the CRM-GAL4 pattern. c Left arrow indicates GAL4 is ectopically expressed in elements of the CRM-LexA pattern. d Trans-repression was seen in this cross (see Figure 5). The results of the transvection test crosses are summa- rized in Table 1. Most importantly, transvection was fre- quently observed. Excluding the three crosses that were GAL4/LexA swaps between CRMs, we found that the trans- vection frequency among unique pairs of CRMs was 17/57 (30%). The fraction of CRM-GAL4s that participated in transvection with any of the three CRM-LexAs was 11/21 (52%). Assuming this collection of CRMs is representative of enhancer fragments found throughout the genome, we con- clude that a large fraction of regulatory DNA is able to sup- port transvection if removed from its endogenous locus and used to drive the expression of a transgene. Transvection and its directionality requires compatibility between CRMs For the 10/21 CRM-GAL4s that showed no evidence of transvection, it might be that sequence elements within those CRMs prevent transvection from occurring. However, for the 11 CRM-GAL4s that did show transvection, six of them showed transvection with at least one, but not all of the CRM-LexAs that we tested. This indicates that there are differences in transvection compatibility between different pairs of CRMs and suggests that there are sequences within the CRMs that encode these compatibilities. Thus it may be that those CRM-GAL4s that showed no transvection are simply incompatible with the three CRM-LexAs that we used for testing. One interesting ﬁnding from the 60 test crosses is that transvection can be unidirectional between transgenes that Table 1 Summary of transvection occurring in test crosses to three CRM-LexA strains GAL4 used R24B02 R23G07 R58G08 )[ )[ )[ ) — — ) — — [ — — — — — — — — — — use the same core promoter (DSCP), but different CRMs. Thirteen of the 57 unique pairs of CRMs showed unidirec- tional transvection. This directionality is also dependent on compatibility between CRM pairs. For example, trans- vection with R24B02-GAL4 was bidirectional when it was paired with R58G08-LexA, but unidirectional when it was paired with R23G07-LexA, as features of the R24B02 pattern showed up with the LexAop reporter but no element of the R23G07 pattern showed up with the UAS reporter. This directionality was not due to differences between the GAL4/LexA systems, because swapping GAL4/LexA be- tween R24B02 and R23G07 had no effect on the di- rectionality between the CRMs (Table 1). Transvection does not always occur in all cells For many of the combinations of the CRM transgenes we examined, transvection only occurred in a subset of cell types in which the transgenes were expressed. One example of this comes from the R24B02/R58G08 pair, which exhibited bidirectional transvection. Though R24B02 is able to drive expression in trans in hemilineage 12A in segments S2 to T2 (Figure 4, A and B), there is very little transvection in 12A in segment A1 (Figure 4, A and C). R24B02 also drives extensive expression in neurons in the optic lobes, but also fails to par- ticipate in transvection in these cells (Figure 4D). This pattern of cell type speciﬁcity is consistent between individuals and does not differ between integration sites, because the pattern of cells showing transvection between R24B02/R58G08 is the same regardless of whether the transgenes are inserted in attP40 or attP2 (data not shown). The consistency of the pattern of cell type speciﬁcity suggests that it is due to sequen- ces within the CRMs. We also saw several cases among our 60 test crosses where transvection was sparse and/or stochas- tic (data not shown). We conclude from these observations that the factors affecting transvection must differ between cell types. GAL4 bound to UAS can activate transcription in trans The observation that transvection occurs only between compatible pairs of CRMs led us to consider the possibility that there are sequences in the CRMs that regulate trans- vection. Compatibility between CRMs could then be viewed as compatibility between binding sites for one or more transvection-mediating factors. To ask whether there are sequences that confer the ability of an enhancer to activate transcription in trans, we examined the effect of pairing R24B02-LexA with pJFRC2-10XUAS-IVS-mCD8::GFP (here- after referred to as UAS-mCD8::GFP). The UAS-mCD8::GFP transgene contains no regulatory DNA other than GAL4 binding (UAS) sites and the hsp70 basal promoter (Brand and Perrimon 1993; Pfeiffer et al. 2010); thus it is a minimal construct that should not contain any of the sequences that would be present in endogenous Drosophila enhancers that may uniquely confer the ability to act in trans. When we examined larvae that had UAS-mCD8::GFP paired with R24B02-LexA in attP2, we found that driving Transvection at phiC31 Integration Sites 1135