been observed at both of these gene loci (Jack and Judd 1979; Gelbart and Wu 1982; Geyer et al. 1990), it remains a possibility that there are sequences carried in the trans- genic constructs used to insert the attP sites and CRM trans- genes that promote or are necessary for transvection. One solution to this problem would be to use recombinase- mediated cassette exchange (Bateman et al. 2006) to cleanly integrate transgenic constructs without inserting dominant markers. We also ﬁnd that transvection does not always occur throughout the entire expression pattern, suggesting some element of cell-type speciﬁcity. This observation may reﬂect a difference in the degree of somatic pairing between cell types. We favor a second possibility: that distinct elements of an expression pattern depend on unique combinations of transcription factors, and these transcription factor combi- nations differ in their ability to activate the trans promoter. Thus, the cell speciﬁcity of transvection and compatibility differences between CRMs may be due to the same under- lying mechanism. Trans-repression and a possible role for Polycomb group proteins Surprisingly, we ﬁnd evidence that the epigenetic state of a transgene can be transmitted in trans in Drosophila. Trans- repression and transmission of an epigenetic state has been observed not only in Drosophila (Henikoff and Dreesen 1989), but also plants (reviewed in Chandler 2007) and mammals (Rassoulzadegan et al. 2002), so epigenetic trans- repression may be a common feature of genome function. The phiC31 system could be used with deﬁned CRM transgenes to work out mechanisms of trans-repression in the same way it can be used to study trans-activation, providing insight into this feature of gene regulation that may be applicable across diverse species. One explanation for the trans-repression that we describe here is that there are sequences within R23G07 that recruit chromatin-silencing proteins, and these chromatin silencers produce chromatin modiﬁcations on both transgenes that are inherited and maintained by the neuroblast progeny (Figure 5E; model 1). Alternatively, the trans-repression we observe may not require both CRMs to be epigenetically mod- iﬁed. Instead, the transgene that is modiﬁed may persistently repress its paired, unmodiﬁed transgene (Figure 5E; model 2). We do not know how common trans-repression may be, be- cause it is much more difﬁcult to detect than positive trans interactions using the transgene crossing scheme that we used. However we expect that the frequency we observed (1 out of 57 crosses) is a low estimate. Though we do not know the mechanism of silencing that occurs with R23G07, one attractive possibility is that it is regulated by Polycomb group proteins (reviewed in Mateos- Langerak and Cavalli 2008; Simon and Kingston 2009). In support of this, the region of the genome containing the R23G07 fragment has been found to bind Polycomb group (PcG) proteins Pho, Pcl, Pc, Psc, and dSfmbt in embryos and derived cell lines (Oktaba et al. 2008; Celniker et al. 2009). Moreover, R23G07 contains at least one sequence, CCGGCCATTCG, that is a reasonable match (8/11 nucleo- tides) for a position sequence-speciﬁc matrix reconstructed from ChiP-chip analysis of Pho and dSfmbt binding (G[C/A] [C/G]GCCAT[T/C]TT; Oktaba et al. 2008). Given that PcG proteins are involved in pairing-dependent silencing (Kassis et al. 1991; Brown et al. 1998), it would be interesting to determine whether they are involved in the trans-repression we have reported here. If so, the phiC31 system could also be a powerful tool for dissecting the mechanism that under- lies the trans effects of PcG-dependent gene silencing. Implications for transgene usage in Drosophila We ﬁrst encountered transvection while attempting to independently label and manipulate multiple classes of neurons (R. Harris, personal communication). This goal requires transgenes to function independently. Indepen- dence in the transcriptional regulation of transgenes is particularly important for intersectional strategies, whereby the expression patterns of two or more transgenes are subdivided or combined. For example, the split-GAL4 system (Luan et al. 2006) is often used to target cells that are in common between two expression patterns. The DNA bind- ing domain of GAL4 is expressed in one pattern (CRM1- GAL4DBD), the activation domain of GAL4 is expressed in a second pattern (CRM2-GAL4AD), and only in the inter- section of the two patterns does the reconstituted GAL4 protein drive expression of UAS transgenes. The lack of in- dependence in the transcriptional regulation of each split GAL4 component could result in an unexpected pattern. For example, transvection between CRM1-GAL4AD and CRM2-GAL4DBD could broaden the expression domain of CRM2-GAL4DBD to include more CRM1-GAL4AD express- ing cells than expected. This could lead to an experimental result being misinterpreted. Because of the apparent widespread nature of trans- vection between transgenes inserted at integration sites, caution should be exercised when using these genetic tools. To minimize the confounding effects of transvection, we propose the following guidelines: (1) Wherever possible, the use of integrations sites should be limited to one insertion per site, per genotype. We have seen no case of transvection between transgenes inserted into the genome randomly by transposon-based methods or transvection between two transgenes inserted at distant sites. (2) If the same site must be used for two different transgenes, those two transgenes must not require in- dependence. For example, there should be no problem with using a CRM-GAL4 and UAS-GFP in the same site, because there is an intended activation interaction between those two transgenes. (3) If two transgenes must be independent in their transcriptional control and also inserted into the same genomic site, controls must be in place to test for possible Transvection at phiC31 Integration Sites 1139