We next examined a genotype in which both R58G08- GAL4 and R24B02-LexA were integrated into attP40, restor- ing the ability of the two transgenes to pair at a location distant from attP2. This genotype exhibited the same pat- tern of transvection that we had previously observed with the attP2 insertions (Figure 2E). Because the strength and pattern of transvection for the paired CRM transgenes was qualitatively similar between attP2 and attP40, we also con- clude that there is no signiﬁcant position effect on trans- vection, at least with respect to these two sites. We did not examine other attP sites, so it is possible that there may be regions in the genome that prevent transvection and that some attP sites may lie within those regions. However, pre- vious work from others indicates that the genome is generally permissive for transvection (Chen et al. 2002), and our results with attP40 and attP2 are consistent with that view. Promoter competition inﬂuences transvection directionality between transgenes We next asked whether the type of promoter used in the CRM transgenes inﬂuences transvection. To test whether the DSCP (Pfeiffer et al. 2008) used in the construction of the initial CRM-GAL4 library is required for transvection effects, we replaced it with the P-element transposase gene promoter (O’Hare and Rubin 1983) in two CRM transgenes to create R58G08-Ptrans-GAL4 and R24B02-Ptrans-GAL4. When examined individually, both P-transposase promoter transgenes expressed in the neuronal hemilineages in which they expressed when their CRMs were combined with the DSCP, but at reduced levels (data not shown), consistent with previous observations that the P-transposase promoter is a weaker promoter than the DSCP for some CRM trans- genes (Pfeiffer et al. 2008; B. Pfeiffer, personal communica- tion). Both P-transposase promoter transgenes were also expressed in cells that are not found in the expression pat- terns of the DSCP transgenes. Thus the pattern and strength of expression driven by the R58G08 and R24B02 CRMs are partially dependent on the identity of the core promoter. Having established that the CRM-Ptrans-GAL4 transgenes are each expressed in the same hemilineage as the original CRM-DSCP-GAL4 transgenes, we next tested for transvec- tion between R58G08-Ptrans-GAL4 and R24B02-LexA when both were inserted in attP2. We also tested the complemen- tary pairing between R24B02-Ptrans-GAL4 and R58G08- LexA in attP2. In both cases, the CRM of the transgene utilizing the P-transposase promoter activated transcription in trans, whereas the CRM of the transgene utilizing the DSCP did not (Figure 3, A and B). This unidirectional trans- vection could be due to one of two reasons. First, it may be that the DSCP can be regulated by the trans CRM, but the P-transposase promoter cannot. The second possibility is that transvection may tend to operate in one direction when there is an imbalance in the strength of the promoters. The DSCP may outcompete the trans P-transposase pro- moter for transcriptional activation by the CRM in cis to the DSCP, leading to the apparent unidirectionality of the transvection effect that we observed. Previous studies have shown that the strength or presence of the cis promoter can inﬂuence whether an enhancer will act in trans (Geyer et al. 1990; Martínez-Laborda et al. 1992; Morris et al. 1999, 2004; Lee and Wu 2006), supporting the latter hypothesis. To test whether the DSCP promoter was responsible for the unidirectional transvection we observed between R58G08-Ptrans-GAL4 and R24B02-LexA, we generated the promoterless construct R24B02-DP-LexA and inserted it into attP2. R24B02-DP-LexA did not drive speciﬁc expression in hemilineage 12A on its own, as expected, but instead pro- duced a low level of nonspeciﬁc neuronal expression (Figure 3E). However, when we paired attP2 insertions of R24B02- DP-LexA and R58G08-Ptrans-GAL4, we found that the R24B02 CRM drove GAL4 expression speciﬁcally in hemi- lineage 12A in trans (Figure 3, E–G). Thus, the P-transposase promoter can be regulated by a trans-acting enhancer. This result excludes the possibility that the unidirectional transvec- tion we observed between CRM-Ptrans-GAL4 and CRM-DSCP- LexA transgenes was due to the insensitivity of the P-transposase promoter to transvection. We conclude that the DSCP can out- compete a weak trans promoter for activation by the CRM in cis to the DSCP, which can affect the apparent directionality of transvection between two transgenes. Transvection between R58G08-LexA and R24B02-GAL4 does not require zeste Some examples of transvection depend on the function of the zeste (z) gene (Kaufman et al. 1973; Gelbart and Wu 1982), although it is not required in all cases (Pattatucci and Kaufman 1991; Hendrickson and Sakonju 1995). To test whether z function is required for transvection between paired transgenes inserted at attP2, we examined transvec- tion between attP2 insertions of R24B02-GAL4 and R58G08- LexA in larvae that were homozygous or hemizygous for za, a putative loss-of-function or strongly hypomorphic allele (Jack and Judd 1979). The pattern of transvection in these z mutant larvae was no different from what was found in larvae wild type for z, indicating that z is not required to mediate transvection between R24B02-GAL4 and R58G08- LexA (Figure 3C). We cannot rule out that the neomorphic mutation z1 might modify the transvection phenotype, as it was not tested. This is important to note because z1 can cause phenotypes that are pairing dependent even when there is no obvious z-null phenotype. The zeste-white inter- action is one such example (Jack and Judd 1979; Gelbart and Wu 1982). Nonetheless, we conclude that z function is not necessary for pairing and transvection between trans- genes inserted at attP2. A large fraction of regulatory DNA can support transvection The example of transvection between transgenes built with the R58G08 and R24B02 CRMs led us to ask whether this effect is speciﬁc to this particular combination of CRMs. To test whether transvection is common between transgenes Transvection at phiC31 Integration Sites 1133