The Capacity to Act in Trans Varies Among Drosophila Enhancers

Abstract

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.

Figure 1

Study strategy. (A) Each enhancer is cloned into a common reporter carrying a minimal hsp70 promoter flanked by loxP sites and upstream of a lacZ coding region. Enhancer activation of the promoter in cis can be assessed by staining for β-gal activity. Enhancer action in trans is assessed by placing each lacZ construct in trans to an enhancerless hsp70-GFP construct at the same genomic location and assessing GFP fluorescence. Note that the promoter of the lacZ construct can also be removed by Cre/loxP-mediated recombination. (B) Cartoon representation of a third instar eye disc. The morphogenetic furrow (MF) represents a wave of mitotic divisions that moves from the posterior (down) to the anterior (up) of the disc. Cells anterior to the MF are precursor cells that have not yet differentiated, while those posterior to the MF initiate a pattern of differentiation to form ommatidial clusters (OC). Each mature cluster consists of eight photoreceptor cells, R1–R8, surrounded by four cone cells (c), all of which develop via a stereotyped developmental program following the passage of the MF. OC schematic is adapted from Mavromatakis and Tomlinson (2013).

Figure 2

Enhancers active in the third instar eye disc act in trans with varying strengths. Each panel shows a representative eye disc carrying the indicated enhancer in trans to hsp70-GFP, with the fraction of scored discs that show GFP-positive cells indicated. The wing-specific enhancer sal was used as a negative control. Each enhancer was qualitatively assigned to a strong, moderate, or weak transvection class based on the number of GFP-positive cells observed (see main text). Arrowheads indicate weak GFP-positive cells.

Figure 3

G-TRACE-based strategy further supports enhancer action in trans by weak class enhancers. (A) Schematic for an alternative detection scheme, where hsp70-GFP (Figure 1A) is replaced with hsp70-FLP. Trans-activation of FLP at suitable levels removes a transcriptional stop signal on a G-TRACE cassette located at a different genomic location, resulting in strong GFP fluorescence in that cell. Schematic adapted from Evans et al. (2009). (B) Scoring of GFP-positive cells using the G-TRACE system. Each symbol represents the number of GFP-positive cells in a single disc; lines overlaying each cluster of symbols represent mean ± SD. (C) Representative eye discs from flies carrying the indicated enhancer activating the G-TRACE system (nmo and aos not shown).

Figure 4

The enhancer ey acts in trans early in development. (A) Eye disc with clonal regions of GFP-positive cells resulting from trans-activation of hsp70-FLP by the ey enhancer and subsequent activation of the G-TRACE cassette. Dashed line, morphogenetic furrow. (B) Z-projected confocal image of inset area from A. Clones (dashed circles) were defined as overlapping/touching groups of GFP-positive cells (>2) with no evidence of intervening GFP-negative cells. Single GFP-positive cells contacting only GFP-negative cells (arrows) were not scored. The region shown was scored as six clonal patches, with between 6 and 24 GFP-positive cells per clone. (C) The number of GFP-positive cells per clone was counted or estimated for 30 clonal patches. Overlaid lines represent mean ± SD.

Figure 5

Cell-type specificity of transvection correlates with strength and timing of cis-activity. (A) Eye discs showing trans-activation of hsp70-GFP by the enhancer GMR. Costaining with anti-Elav shows that the majority of GFP-positive cells are either R3 or R4. (B) Image shows a single ommatidial cluster with GFP driven in cis by GMR. The positions of R2–R4 are highlighted. Relative fluorescent intensities of R4 vs. R2 (blue) or R3 (red) show higher R4 expression relative to R2 (2.17 ± 0.55), but equivalent expression in R4 and R3 (1.06 ± 0.26). (C and D) Eye discs showing trans-activation of hsp70-GFP by sev (C) or fas2 (D) enhancers with costaining for the neuronal marker Elav.

Figure 6

Enhancer transvection class correlates with enhancer strength in cis. (A) β-Gal staining of eye discs from representative strong and weak class enhancers. All discs were stained under identical conditions. (B) Relative levels of lacZ mRNA generated by different enhancers as assessed by quantitative RT-PCR from eye-antennal discs. Levels are normalized to the enhancer ey (= 1.0). Enhancers of the strong transvection class were more likely to drive higher levels of lacZ mRNA in cis.

Figure 7

Multimerization of the pax enhancer increases its capacity to support transvection. (A) Staining of eye discs carrying paxpaxpax-LTL-lacZ with antibodies against β-gal and Elav. Arrowheads show β-gal-positive cells that overlap Elav-positive neurons, whereas arrows show cells with positions consistent with cone cells. Note that the signal for β-gal appears weak at high resolution due to the weak nature of the LTL promoter. (B) Representative eye discs carrying pax or paxpaxpax showing trans-activation of hsp70-GFP. Multimerization of the pax enhancer results in a change from weak to strong transvection class. (C and D) Eye discs showing paxpaxpax activation of hsp70-GFP in trans, with staining for GFP and either the cone cell marker Cut (C) or the neuronal marker Elav (D). Cells showing overlap (arrowheads) and no overlap (arrows) of signals are visible for both markers.

Figure 8

Analysis of wing enhancers further supports a link between enhancer strength and transvection class. (A) β-Gal staining and trans-activation of hsp70-GFP and the hsp70-FLP/G-TRACE system is shown for the wing disc enhancers Ser, ct(2.7), and sal. Ser, the strongest enhancer in cis, shows more consistent and robust trans-action than the weaker ct and sal enhancers. Arrowheads highlight β-gal-positive or GFP-positive cells in the expected domains of the wing pouch for ct and sal; arrows marks the air sac primordium (ASP). The fraction of scored discs that showed GFP-positive cells is given for each test for trans-activation. The ASP was not scored in discs activating hsp70-GFP due to high levels of background fluorescence in that region. (B) Multimerization of sal to create salsalsal increases cis-expression ∼45-fold as assessed by quantitative RT-PCR on wing discs. Data represent two independent experiments. Inset shows β-gal staining of a wing disc carrying salsalsal-LTL-lacZ. (C) salsalsal activates hsp70-GFP in trans at high levels relative to a single copy of the sal enhancer.

Figure 9

Factors beyond enhancer strength may also contribute to transvection. (A) Truncated fragment of the 2.7-kb ct enhancer produces higher levels of lacZ expression in cis as assessed by quantitative RT-PCR. Data represent three independent experiments. (B) β-Gal staining shows the ct(668) enhancer fragment is active at the wing margin. (C) No trans-activation of hsp70-GFP is detected at the wing margin for the shorter ct enhancer fragment.