Academic journal article Genetics

The Role of Insulators in Transgene Transvection in Drosophila

Academic journal article Genetics

The Role of Insulators in Transgene Transvection in Drosophila

Article excerpt

TRANSGENIC reporter genes are powerful tools for studying gene regulation. However, a transgene is often susceptible to interactions with surrounding chromatin, leading to significant variations in expression patterns and levels, depending on the site of insertion in the genome (Levis et al. 1985). In order to factor-out these "position effects," multiple lines of transgenic animals must be studied. Recently, this problem has been tackled by two strategies now widely used in Drosophila. One relies on the ability to guide integration of a transgene to a specific location in the genome via the use of the ФСЭ1 -mediated integration system (Thorpe and Smith 1998; Groth et al. 2004; Markstein et al. 2008; Pfeiffer et al. 2008; Kvon 2015). By targeting all reporters to the same "landing site," they are directly comparable, and only a single transgenic line per construct needs to be analyzed. The second way to minimize genomic position effects utilizes insulator sequences in transgenesis vectors. Insulator DNA elements were first identified by their potential to block gene function when interposed between enhancer and promoter (Kellum and Schedl 1992; Cai and Levine 1995; Kuhn et al. 2003), and to insulate transgenes from effects of surrounding chromatin (Kellum and Schedl 1991; Roseman et al. 1993, 1995). These faculties of insulators seem to derive from their strong propensity for interaction with each other (Kuhn et al. 2003; Chetverina et al. 2008; Kyrchanova et al. 2008b, 2011). Such interactions are proposed to form chromatin loops bridging even distant loci at the base of the loop and isolating interactions inside the loop from interactions outside the loop (Blanton et al. 2003; Byrd and Corces 2003; Doyle et al. 2014; Cubeñas-Potts and Corces 2015).

We started using both of these approaches to characterize enhancer modules of two neighboring Drosophila genes E(spl)m7 and E(spl)m8. To that end we generated a series of reporter constructs flanked by two copies of the insulator sequence from the gypsy transposon (gypsy insulator, GI) and integrated each construct into the same attP locus. When we tested two different reporters in a heterozygous configuration, we noted that they markedly affected each other's expression in trans. The ability of enhancers to activate promoters in the homologous locus is called transvection and was first reported in Drosophila, as a phenomenon of pairing-dependent intragenic (unexpected) complementation in loci like Ultrabithorax (Ubx) (Lewis 1954; Martínez-Laborda et al. 1992), decapentaplegic (dpp) (Gelbart 1982), yellow (Geyer et al. 1990) and white (Babu and Bhat 1980). Subsequent studies employing randomly integrated P-element transgenes showed that the Drosophila genome is generally permissive to enhancer action in trans (Chen et al. 2002; Kravchenko et al. 2005) offering a first glimpse of the molecular basis of this phenomenon and reconfirming the need for somatic homolog pairing (or synapsis). In dip-terans, like Drosophila, homolog synapsis is not limited to germline meiotic cells, but is very common in somatic tissues. This is in contrast to mammals, where somatic homolog synapsis seems to happen only in special occasions, and, accordingly, only sporadic cases of transvection have been reported (McKee 2004; Heride et al. 2010; Apte and Meller 2012; Stratigi et al. 2015; Joyce et al. 2016). The development of site-specific integration methods in Drosophila (such as the FC31 -based recombination method) has recently rekindled the interest in transvection (Lee and Wu 2006; Bateman et al. 2012a; Mellert and Truman 2012; Fujioka et al. 2016). However, the mechanism of transvection is still poorly understood.

Using our series of reporter transgenes, we decided to search for sequence determinants of transvection. We found that this interaction is dependent on the presence of homotypic insulator DNA elements on both homologs. Our transgenesis vectors contained two such insulators: the Gypsy Insulator (GI) commonly utilized to protect transgenes from genomic position effects, and the Wari Insulator (WI) carried in the 3' part of the mini-white marker gene. …

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