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The Drosophila Gypsy Insulator Supports Transvection in the Presence of the Vestigial Enhancer

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The Drosophila Gypsy Insulator Supports Transvection in the Presence of the Vestigial Enhancer The Drosophila gypsy Insulator Supports Transvection in the Presence of the vestigial Enhancer Todd Schoborg☯, Srilalitha Kuruganti¤a☯, Ryan Rickels¤b, Mariano Labrador* Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, United States of America Abstract Though operationally defined as cis-regulatory elements, enhancers can also communicate with promoters on a separate homolog in trans, a mechanism that has been suggested to account for the ability of certain alleles of the same gene to complement one another in a process otherwise known as transvection. This homolog-pairing dependent process is facilitated in Drosophila by chromatin-associated pairing proteins, many of which remain unknown and their mechanism of action uncharacterized. Here we have tested the role of the gypsy chromatin insulator in facilitating pairing and communication between enhancers and promoters in trans using a transgenic eGFP reporter system engineered to allow for targeted deletions in the vestigial Boundary Enhancer (vgBE) and the hsp70 minimal promoter, along with one or two flanking gypsy elements. We found a modest 2.5-3x increase in eGFP reporter levels from homozygotes carrying an intact copy of the reporter on each homolog compared to unpaired hemizygotes, although this behavior was independent of gypsy. However, detectable levels of GFP protein along the DV wing boundary in trans-heterozygotes lacking a single enhancer and promoter was only observed in the presence of two flanking gypsy elements. Our results demonstrate that gypsy can stimulate enhancer-promoter communication in trans throughout the genome in a context-dependent manner, likely through modulation of local chromatin dynamics once pairing has been established by other elements and highlights chromatin structure as the master regulator of this phenomenon. Citation: Schoborg T, Kuruganti S, Rickels R, Labrador M (2013) The Drosophila gypsy Insulator Supports Transvection in the Presence of the vestigial Enhancer. PLoS ONE 8(11): e81331. doi:10.1371/journal.pone.0081331 Editor: Charalampos Babis Spilianakis, University of Crete, Greece Received August 23, 2013; Accepted October 21, 2013; Published November 13, 2013 Copyright: © 2013 Schoborg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank the BCMB Department, the College of Arts and Sciences and Office of Research at the University of Tennessee for support in addition to US Public Health Service Award GM78132-2 from the NIH and MCB-0616081 from NSF to ML. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ☯ These authors contributed equally to this work. ¤a Current address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America ¤b Current address: Stowers Institute For Medical Research, Kansas City, Missouri, United States of America Introduction enhancer and the promoter, mediated by a number of regulatory proteins, general transcription factors, RNA Pol II Unlocking the mechanism by which gene regulatory and chromatin binding proteins that result in the formation of elements (enhancers, promoters and other transcription factor chromatin loop structures that are critical for transcriptional binding sites) coordinate gene expression in a precise spatio- activation [5-9]. temporal manner is critical to understanding how eukaryotic However, the ability of enhancers to also act in trans (i.e., on genomes function in vivo. Chromatin structure plays a central a separate DNA molecule) on promoters has been observed role in this process, where nucleosome position and density, both in vitro and in vivo [7,10-14] where such behavior has chromatin insulators, histone modifications and their associated been suggested to account for a number of homolog pairing- proteins function to modulate the properties of these regulatory dependent phenotypes, such as in the phenomenon of elements. Most of the effort in understanding this interplay has transvection (see 15-19 for review). The term transvection was focused on their behavior in cis—that is, interactions occurring first coined by E.B. Lewis in 1954 to describe the ability of along the same chromosome. Indeed, enhancers are defined certain Drosophila alleles of Ultrabithorax (Ubx) to complement as cis-regulatory elements that function to stimulate gene one another, leading to partial rescue of the mutant phenotype expression when located either distal or proximal to their [20]. Importantly, this rescue failed when the locus on either cognate promoters, and can function over large distances [1-4]. homolog was relocated to a new position on the chromosome, This is achieved through physical association between the suggesting that somatic pairing between homologous PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e81331 Chromatin Insulators and Transvection chromosomes is essential for transvection. This type of retrotransposon is perhaps the most well characterized intragenic complementation has been reported almost insulator, whose enhancer and heterochromatin blocking exclusively in Drosophila, with transvection effects observed at properties are conferred by Su(Hw), CP190 and a number of loci: yellow (y) [21,22], decapentaplegic (dpp) [23], Mod(mdg4)67.2. However, in addition to gypsy there are eyes absent (eya) [24], white (w) [25], Gpdh [26], hedgehog thousands of endogenous insulator sites located throughout (hh) [27], wingless (wg) [28], engrailed (en) [29], pointed (pnt) the genome, where combinatorial binding of insulator proteins [30], cubitus interrruptus (ci) [31], sex combs reduced (scr) [32], to each of these sites suggests a complex landscape whose brown (bw) [33,34] and vestigial (vg) [35]. Recent studies have functional consequences remain poorly understood [54-56]. concluded that transvection is pervasive throughout the entire Recent work has shown that these elements help mediate Drosophila genome [11,36], a finding not surprising given that long-range contacts between enhancers, promoters and other homologs remain paired in somatic nuclei during interphase in insulator sites in order to direct transcriptional outputs, maintain flies [37]. In other eukaryotes, homologs in somatic tissue do regions of histone modifications, and establish gene regulatory not remain in close synapse throughout interphase, yet quite a and physical domains [2,57-60]. few cases of transvection and other pairing dependent Though most of these interactions are thought to occur in cis, phenomenon have been reported in model systems other than potential interactions in trans are not out of the question, even Drosophila, including yeast [38], plants [39] and mammals for long range contacts between non-homologous sites, [40-42], suggesting that eukaryotes possess evolutionarily analogous to the behavior observed for olfactory receptor conserved mechanisms that allow homologous chromosomes choice by the H enhancer and various olfactory gene to communicate in trans. promoters located on different chromosomes in mice [61]. Given the need for physical associations between enhancers Interestingly, gypsy insulators have been previously implicated and promoters to generate a sustained transcriptional output, in transvection, both directly and indirectly, at the yellow locus homolog pairing in trans can facilitate contacts between a [21,62]. Additionally, the bx34e allele, used by E.B. Lewis in his functional enhancer located on one homolog and a functional original description of the phenomenon [20], results from a promoter located on the other, increasing the frequency of gypsy retrotransposon insertion between the bx enhancer and collisions between the two elements and thus the probability the promoter of Ubx and subsequent loss of enhancer- that a stable ternary complex is established. A number of promoter communication in cis due to the enhancer blocking proteins have been shown to be required for homolog pairing in property of the gypsy insulator. Furthermore, reduction of Drosophila meiosis, including the multi-subunit cohesin Su(Hw) has been shown to reduce somatic pairing by ~30% in complex (SMC1, SMC3, SCC1/RAD21, SCC3), although it embryos [63], suggesting that insulators might contribute to does not appear to be required for somatic paring [43]. In pairing dependent enhancer-promoter communication in trans. mammals, this complex has been shown to be involved in Here we have used a reporter construct designed to stable cis looping contacts between enhancers and promoters, elucidate the role of the gypsy insulator in transvection. We suggesting it may play a more direct role in gene regulation engineered our system with the vestigial (vg) boundary [44-46]. Only a handful of Drosophila genes, mainly involved in enhancer (vgBE) and a minimal hsp70 promoter to drive eGFP mitotic functions, cell cycle control and chromatin organization, expression, flanked by a gypsy insulator upstream and including Topoisomerase II (Top2) have been shown to downstream (2-insulator), upstream only (1-insulator) or no promote somatic pairing [43,47], although other chromatin gypsy insulator
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