The interphase nucleus is organized such that genomic segments interact in cis, on the same chromosome, and in trans, between different chromosomes. In Drosophila and other Dipterans, extensive interactions are observed between homologous chromosomes, which can permit enhancers and promoters to communicate in trans. Enhancer action in trans has been observed for a handful of genes in Drosophila, but it is as yet unclear whether this is a general property of all enhancers or specific to a few. Here, we test a collection of well-characterized enhancers for the capacity to act in trans. Specifically, we tested 18 enhancers that are active in either the eye or wing disc of third instar Drosophila larvae and, using two different assays, found evidence that each enhancer can act in trans. However, the degree to which trans-action was supported varied greatly between enhancers. Quantitative analysis of enhancer activity supports a model wherein an enhancer's strength of transcriptional activation is a major determinant of its ability to act in trans, but that additional factors may also contribute to an enhancer's trans-activity. In sum, our data suggest that a capacity to activate a promoter on a paired chromosome is common among Drosophila enhancers.KEYWORDS RMCE; interchromosomal interactions; long-range enhancer; somatic homolog pairing; transvection T HE spatial organization of the interphase eukaryotic genome is characterized by extensive long-distance interactions between distal chromosome regions (Sanyal et al. 2012). Interactions have been identified between sequences on the same chromosome (in cis) or on different chromosomes (in trans) (Lieberman-Aiden et al. 2009;Duan et al. 2010;Sexton et al. 2012;van de Werken et al. 2012;Nagano et al. 2013;Zhang et al. 2013). Many long-distance interactions in cis underlie the activation of specific genes, and in some cases, sequences have been identified that facilitate interactions between a distal enhancer and a specific promoter target (Zhou and Levine 1999;Calhoun et al. 2002;Calhoun and Levine 2003;Lin 2003;Akbari et al. 2008;Fujioka et al. 2009;Majumder et al. 2015). In contrast, the genetic impacts of trans-interactions between chromosomes are less clearly understood. Examples of gene regulation involving interchromosomal associations have been described (Spilianakis et al. 2005;Bacher et al. 2006;Xu et al. 2006;Apostolou and Thanos 2008;Sandhu et al. 2009;Markenscoff-Papadimitriou et al. 2014;Patel et al. 2014), but it remains unclear whether it is common for sequences that regulate gene expression to communicate between different chromosomes when they are physically juxtaposed.In Drosophila melanogaster, extensive trans-interactions are observed between homologous chromosomes in virtually all somatic tissues, a phenomenon known as somatic homolog pairing (reviewed by McKee 2004;Bosco 2012). The close proximity of homologous chromosomes in Drosophila can permit an enhancer to act in trans on a promoter on the paired homolog, a form of pairing-dependent ...