The Role of Insulators in Transgene Transvection in Drosophila

Abstract

Precise activity of a gene requires its promoter to be matched with an appropriate enhancer. Insulators are DNA elements which can limit inappropriate enhancer-promoter interactions. More recently, their ability to stimulate gene activity has been also recognized... Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologs are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologs. Homotypic trans-interactions between four different insulators can support transvection: the gypsy insulator (GI), Wari, Fab-8 and 1A2; GI and Fab-8 are more effective than Wari or 1A2. We show that, in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homolog pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis, and it is further suppressed by cis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans-interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions.

Characterization of Button Loci that Promote Homologous Chromosome Pairing and Cell-Type-Specific Interchromosomal Gene Regulation

“…Our data clearly indicate that transvection can also be disrupted by the loss of small DNA elements, even when pairing is preserved. Consistent with our findings using endogenous alleles, insulators play roles in transvection between transgenes (Lim et al, 2018;Piwko et al, 2019).…”

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila

“…studies independently found enrichment for DNA-binding architectural and insulator proteins in tight-pairing regions (AlHajRowley et al 2019), suggesting a potential role for these proteins in button function. In support of this view,Viets et al observed that genomic regions amenable to pairing are enriched in clusters of insulator proteins(Viets et al 2019), and previous works on the incorporation of insulator sequences into transgenes showed that these sequences can stabilize pairing and transvection(Lim et al 2018;Fujioka et al 2016;Piwko et al 2019). Notably, our analysis revealed a requirement for some degree of specificity between homologous buttons (Figure2-figure supplement 4A,B), since simulations of non-specific interactions between buttons did not result in robust pairing (Fig.…”

Highly structured homolog pairing reflects functional organization of the Drosophila genome

“…41 ), the observation that some insulator proteins and insulator elements promote transvection (refs. 16,19,42,43 , and reviewed by ref. 44 ), and the enrichment of architectural proteins at genomic sites involved in allelic interactions 27 .…”